不同填充材料及脂肪源干细胞移植对骨质疏松大鼠的影响
|
摘要
背景
骨质疏松主要的特征是骨量减少和骨微结构破坏,导致骨脆性增加同时易于骨折的代谢性疾病。随着老龄化社会的发展,骨质疏松患者不断增多,如何有效预防和治疗骨质疏松已经成为国内外的热门话题。根据原林教授提出的“筋膜学说”：人体可以看做是由尚未分化的非特异性结缔组织(筋膜)支架构成的支持与储备系统与被该支架支持与包绕的已分化为各种功能细胞构成的功能系统共同组成。建立在各种功能细胞的专能特化的基础上的功能系统是被以筋膜支架中未分化的干细胞为核心的支持与储备系统支持和包绕的。支持与储备系统内未分化干细胞在微环境下通过分化为各种定向干细胞,源源不断的为功能系统中各种功能细胞的更新提供细胞源泉,同时为功能系统中的各种功能细胞的代谢提供营养,维持其内环境的稳定。因此,在支持与储备系统的支持下功能系统才可能维持结构和功能的稳定,当某种功能细胞在代谢过程中不能及时得到新细胞的补充,出现代谢障碍,机体就可能会出现相应的病变。骨质疏松的发病机制正是在骨组织吸收后,筋膜结缔组织支架中未分化的干细胞未能充分向成骨细胞分化,造成骨形成不足而出现的一种疾病。因此,如何干预骨代谢过程中出现的负平衡,如何有效保证并增加筋膜结缔组织支架中未分化的干细胞,使成骨细胞得到有效的补充,促进骨的形成,重建骨的正平衡是治疗骨质疏松的关键。
目前,骨缺损的修复仍是骨科临床中的棘手难题之一,以往研究显示采用骨移植治疗骨缺损的方法取得了一定的效果,但此方法存在较大的局限性。自体骨移植一直以来都被认为是骨缺损修复的一个金标准,由于植取骨量有限,已形成供区畸形,取骨手术也同样增加了患者的创伤痛苦；异体骨移植虽然取材方便,易获取,可减少手术次数,但是由于其具有抗原性,可引起机体的免疫排斥反应,同时存在着潜在传播疾病的风险,故异体骨移植的并发症也较多,如感染、骨折、骨不愈合等等。因此,人们对新型移植替代物的需求也不断增加。目前各种骨水泥已经备受青睐。伴随人口老龄化的发展,骨质疏松症已成为威胁着中老年人健康的主要疾病。骨质疏松极易并发骨折,同时这骨折也常伴有松质骨压缩及骨缺损。伴随着骨质疏松绝对人口的增加,因各种原因引起的骨缺损合并骨质疏松的患者也逐年增加。当机体骨质疏松时,由于破骨细胞活性增强,骨的吸收大于骨的形成,故新骨生成能力下降,导致骨质疏松骨缺损的愈合时间明显延迟,而新生骨痂减少,愈合后的骨生物力学强度差,容易骨折。与常规骨缺损的治疗相比,治疗骨质疏松骨缺损的花费要更大且传统方法疗效欠佳。骨水泥已逐渐被应用到骨质疏松骨折骨缺损的辅助治疗中,它一方而可增强固定螺钉,防止骨质疏松骨折内固定的松动,另一方面填充骨折的缺损,使其复位及愈合更好,减少发生骨折后遗症。磷酸钙骨水泥近几年也被应用于椎体成形术来治疗骨质疏松椎体压缩骨折,且疗效良好。缓释性骨长因子也渐渐被应用于骨质疏松骨缺损的治疗中,这显著加速了骨缺损的愈合,还在一定程度上改善局部的骨质疏松,增强局部骨生物力学,降低骨折的发生几率。
脂肪组织同样也是两系统理论中的支持与储备系统中重要组成部分,其分离出的脂肪源干细胞(adipose-derived stem cells, ADSCs)是筋膜结缔组织支架中未分化干细胞的一种重要储备形式。脂肪源干细胞的研究逐渐成为干细胞研究的热点。脂肪源干细胞易于获取,便于分离培养；具有多向分化潜能,能跨胚层分化为三胚层内的组织细胞,在特定的环境中可以向成骨、成软骨、成脂、成肌和成神经等诱导分化：具有免疫原性低和调节免疫功能。此外,骨髓间充质干细胞相比,脂肪源干细胞来源更广泛,获取更容易,对于病人而言,带来的创伤和痛苦更小。这些特征为脂肪源干细胞的同体及异体移植提供了更有利条件。因此,通过增加机体筋膜结缔组织内的未分化的脂肪源干细胞,促进其成骨分化,增加骨形成,可能会对骨质疏松起到预防和治疗作用。
利用糖皮质激素建立骨质疏松模型是目前国际上比较公认的骨质疏松症药物模型。长期注射大剂量的糖皮质激素可引起体内钙磷代谢的紊乱,促进甲状旁腺素的分泌,抑制骨保护素的分泌,抑制成骨细胞增殖和活性,增加破骨细胞数量和活性等多种途径促使骨形成减少,骨吸收增加,从而导致骨质疏松的发生。
为了探讨异体脂肪源干细胞移植及不同骨填充材料对骨质疏松的预防和治疗作用,本实验通过观察异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠体内血清钙及磷代谢、骨密度、骨生物力学及骨组织形态计量学等方面的影响进行综合评价,同时通过观察不同填充材料对去卵巢骨质疏松大鼠的骨切片的影响,探索一种新的骨质疏松治疗方法,为临床骨质疏松的预防和治疗提供新的研究思路。
目的
1、体外分离培养腹股沟皮下脂肪源干细胞,并对其进行多向分化能力和表面标志的鉴定,以判断在支持与储备系统中是否存在未分化的脂肪源干细胞；
2、通过对骨密度检测,判断长期给予糖皮质激素是否成功模拟建立骨质疏松模型?
3、通过检测大鼠钙、磷、碱性磷酸酶,观察异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠的骨体内钙、磷代谢和骨形成的影响；
4、通过检测实验大鼠的骨密度,观察异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠骨质量的影响；
5、通过检测三点弯曲试验,观察移植异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠的骨强度和骨刚度的影响；
6、通过对骨组织形态计量学观察,探索异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠骨组织微环境的影响；
7、为进一步证实“筋膜学说”中“通过动员支持与储备系统中未分化干细胞可以治疗退行性和衰老性疾病”提供部分实验依据。
8、通过不同骨填充材料对去卵巢大鼠进行填充后进行相应骨形态学观察,筛选最佳骨材料,为临床新材料的应用提供部分依据。方法
1、取成年SD大鼠腹股沟皮下脂肪垫,I型胶原酶消化,体外分离培养筋膜结缔组织中脂肪源干细胞。分别通过形态学、成脂成骨诱导分化及利用流式细胞术对表面抗原CD11b、CD29、CD45、CD49d、CD90和CD106进行鉴定4代脂肪源干细胞,判断所培养的细胞是否为具有多向分化潜能的脂肪源干细胞。
2、40只SD雌性大鼠随机分成空白对照组(A组)、模型组(B组)、治疗组(C)和治疗对照组(D组)。B、C和D组分别给予颈背部皮下注射醋酸泼尼松龙注射液8mg/kg,3次/周；A组给予颈背部皮下注射生理盐水0.8mL,3次/周。12周后,将A组和B组处死取材,判断造模是否成功。
3、模拟骨质疏松模型成功后,收集传至第4代的脂肪源干细胞,经尾静脉向C组大鼠每只注射脂肪源干细胞3×106个,D组大鼠每只注射1mL生理盐水,4周后处死取材。
4、应用水合氯醛麻醉大鼠后,用促凝管接取心脏血液,室温下放置8h后3000r/min,离心15min后制备血清,分装后-70℃保存。全自动生化分析仪分别利用偶氮砷Ⅲ法、磷钼酸比色法及速率法测定血清中钙、磷和碱性磷酸酶的含量。取血完毕后,采用心脏放血处死,分别取出大鼠双侧股骨和第三至第五腰椎,去除周围肌肉组织。利用双能X线骨密度仪小动物测量软件测定腰椎和右侧股骨的骨密度值。利用BOSE生物材料动态力学试验机对左侧股骨进行三点弯曲实验,股骨背侧向上,腹侧向下,跨距为18mm,速率为0.05mm/s,股骨压断时记录最大载荷和刚度值。
5、处死大鼠后取出右侧胫骨上1/3部分,手术刀片矢状面剖开,固定后酒精逐级脱水,分别浸入浸液I(甲基丙烯酸甲酯70mL+邻苯二甲酸二丁酯30mL)、浸液Ⅱ(浸液I+过氧化苯甲酰1g)和浸液Ⅳ(浸液Ⅰ+过氧化苯甲酰2.5g)各2d,再用新鲜配制的浸液Ⅲ进行包埋。Jung K重型切骨机连续切取6μm厚度切片,分别进行组织学观察(Gemisa染色)和类骨质观察(von Kossa染色)。测量区域为生长板下1mm至4mm范围内,采用Lecia QWin多功能彩色病理图像分析软件进行骨组织形态计量学分析。具体参数包括骨小梁相对体积(BV/TV)X骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)和骨小梁分离度(Tb.Sp)和单位骨小梁破骨细胞数(OC.N),均采用国际通用的公式进行计算。
6、骨质疏松模型手术：正常雌性大鼠全麻成功后,腹部剃毛,碘伏消毒。下腹正中切口,逐层进入腹腔。找出一侧卵巢结扎切除。同法切除另一侧。缝合腹膜及腹肌,最后缝合皮肤。一周拆线。3个月后即为骨质疏松模型。
7、待模型建立成功后,做材垂直骨面钻孔深5毫米建立骨隧道。于隧道内植入材料并轻轻压实至骨面,然后用骨蜡封堵料植入手术：全麻成功后双下肢膝周前内侧剃毛。碘伏消毒。从膝部沿胫骨前棘切口长约2cm。逐层进达骨膜下,骨膜下剥离显露胫骨近端前内侧面,用直径3毫米钻头。止血,冲洗,选取四种生物材料： β-磷酸三钙(p-TCP);硅酸钙(CS),降解后,释放有益元素硅；掺锶硅酸钙(Sr-CS),降解后,释放有益元素硅、锶；45s5生化陶瓷玻璃进行填充骨缺损。缝合伤口。9周后处死取材。
8、应用HE染色、Giemsa、T-blue、Van gieson、番红O&固绿进行染色。观察不同填充材料骨切片形态学变化。
结果
1、经光学显微镜观察,原代培养的ADSCs在24h后大部分可贴壁,细胞呈梭形、圆形、多边形等。细胞长势良好,7d后可以1：3比例传代,之后每3d传一次代。经换液传代至第4代,可见细胞形态均一,呈纺锤形或者多角形。体外可以向成脂、成骨诱导分化。流式细胞术检测发现CD29和CD90呈强阳性,CD106呈弱阳性,CD11b、CD45和CD49d呈阴性。
2、长期大剂量给予糖皮质激素后,与空白对照组大鼠血清钙(3.63±0.20)、血清磷水平(3.12±0.83)和碱性磷酸酶水平(203.8±40.36)、腰椎骨密度(162.70±14.77)、股骨骨密度(254.40±28.95)、骨强度(98.22±10.72)和骨刚度(192.66±42.28)比较,模型组大鼠血清钙(2.60±0.18)和碱性磷酸酶水平(149.50±22.21)、腰椎骨密度(115.70±16.87)、股骨骨密度(183.40±12.28)、骨强度(58.88±12.53)和骨刚度(127.53±19.13)均显著降低,而血清磷水平(5.25±1.11)显著上升(P<0.05)；骨小梁变细、变薄,骨小梁排列紊乱,骨小梁间出现断裂,间距增大,出现陷窝；骨小梁的数量(4.95±0.75)、相对体积(17.31±3.66)和厚度(35.82±3.88)均显著降低(P<0.05),而骨小梁的分离度(171.30±29.72)增高,单位骨小梁破骨细胞数(52.70±10.34)增多(P<0.05)。
3、异体移植脂肪源干细胞后,与模型组比较,治疗组大鼠血清钙(3.01±0.30)和碱性磷酸酶水平(202.5±40.82)、股骨骨密度(226.50±25.54)、(腰椎骨密度149.30±12.52)、骨强度(80.37±9.37)和骨刚度(187.10±30.06)均显著增加,而血清磷水平(2.84±1.78)显著降低(P<0.05)；骨小梁数目增多,骨小梁变厚变粗,骨小梁间出现连接,陷窝减少；骨小梁的数量(6.42±1.24)、相当体积(25.00±4.88)和厚度(39.63±4.16)显著增加,而骨小梁分离度(124.72±29.79)显著降低,单位骨小梁破骨细胞数(29.52±8.38)减少(P<0.05)。
4、各种染色结果显示p-磷酸三钙组9w无炎症反应；而CS硅酸钙和Sr-CS掺锶硅酸钙：相对TCP有较多的成骨,但9w仍有炎症反应。结论
1、两系统理论中的支持与储备系统中含有脂肪源干细胞,并且这种干细胞是一种未分化的间充质干细胞；
2、长期给予大剂量的糖皮质激素可以模拟骨质疏松模型；
3、通过移植异体脂肪源干细胞可有效改善体内钙、磷代谢,增加骨形成,有效预防和治疗骨质疏松,为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法；
4、对“筋膜学说”中“通过动员筋膜结缔组织中储备的未分化干细胞可治疗退行性疾病”提供了部分实验依据。
5、不同的骨填充材料,对骨创伤面影响不同。新材料CS和Sr均可促进骨形成。为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法。
综上所述,移植异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠有一定的治疗及预防作用,能够促进骨形成,增加骨密度,改善骨生物力学性能,增强骨强度,降低骨折的发生率。本实验进一步提示在骨质疏松的研究中应该更加关注提高机体干细胞的数量和促进干细胞向成骨细胞分化及骨组织的形成方面,为临床上治疗骨质疏松提供了一种新的研究思路。同时,不同填充骨质疏松骨缺损的新材料填充后骨切片对比,新材料CS和Sr均可促进骨形成。为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法。
Background:
Osteoporosis is a metabolic disease that characteristic by osteopenia and bone microstructural destroy, which could cause fragility increasing and able to fractured. Due to the increasing osteoporosis patients, research that prevent and treat osteoporosis have an important clinical significance. Recently, Prof. L. Yuan had put forward a new hypothesis, fasciology hypothesis. In fasciology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of is constituted of undifferentiated cells from the network of unspecialized connective tissues (fascia network). The other one is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. Undifferentiated stem cells in the supporting-storing system incessantly migrate to target areas, differentiate into committed-stem cells, and further differentiate into functional cells. The supporting-storing system provides energy and cell reserve for the functional system. The structures and functions of an organism are maintained by the incessant supplement and refreshment from the supporting-storing system to the functional system. Meanwhile, under the regulation of the nervous system and immune system, the fascia network throughout the body regulates the functional and living status of cells and provides a stable environment for the survival of functional cells.
The repair of bone defects is one of the common difficult problem in clinical department of orthopedics, the bone transplantation in the treatment of bone defect has obtained the certain effect, but this method has limitation. Autogenous bone transplantation has been considered the gold standard for the repair of bone defect, but the bone graft is limited, can cause donor deformity, bone operation also additional trauma and anguish; allogeneic bone transplantation can reduce the times of operation, convenient, but because of its antigenicity, can cause immune rejection at the same time, there is the potential spread of disease risk, and allograft bone complications are more, such as infection, fracture and nonunion. Therefore, the demand for the new substitute transplantation increasing. Bone cement has favored. With the increasing of aging population, osteoporosis has become a major disease that threatens the health of middle-aged and elderly people, osteoporosis is very easy to cause fracture, the fractures are often accompanied with cancellous bone compression and bone defects, in addition with the increase of osteoporosis absolute population, due to various causes of patients with bone defect with osteoporosis caused by more and more. Under circumstance of osteoporosis, enhance the activity of osteoclasts, bone resorption and bone formation, decrease new bone formation ability, bone defect healing was delayed, the new callus biomechanical strength reduction, bone healing after the poor fracture easily, therefore, compared with the conventional bone defect, bone defect costly and traditional methods poor efficacy. Auxiliary treatment of bone cement is gradually applied in osteoporotic fracture bone defect, it can enhance the fixing screw pull-out strength, prevent osteoporosis fracture internal fixation loosening, filling the bone defect, make better fracture reduction and healing, reduce the occurrence of sequelae of fracture, in recent years, calcium phosphate cement was used in vertebroplasty surgery for the treatment of osteoporotic vertebral compression fractures, and achieved good effect. Treatment of sustained-release bone length factor has gradually been applied in osteoporotic bone defects, which not only significantly accelerated the healing of bone defects, but also can improve the local osteoporosis in a certain extent, enhancing local bone biomechanics, to reduce the incidence of fracture.
According to fasciology, the process of a life is just that of continuous consumption of the supporting-storing system. The supplementary of functional cells to repair the functions of damaged tissues ensures normal activities of the functional system. Once one of the functional cells could not be updated by the supporting-storing system in metabolism, the body will be disorder. Osteoporosis is the disease that the osteoblasts, supplemented from the undifferentiated stem cells in the supporting-storing system, is insufficient after bone resorption caused by many factors, e.g. menopause, age and glucocorticoid et al. Therefore, when negative balance exists in bone metabolism, how to increase the undifferentiated stem cells in the supporting-storing system to differentiate into osteoblasts may be an effective treatment for osteoporosis.
Adipose-derived stem cells (ADSCs), which isolated from adipose tissue, are main undifferentiated cells in the supporting-storing system. Currently, ADSCs have become one of the focuses in stem cell research. They are easy to be cultured, rich in source and have differentiation of multipotent, even low in immunity. They also have the capacity of cross-mesodermal differentiation, such as osteogenic, chondrogenic, adipogenic, and myogenic et al. Furthermore, ADSCs are isolated more easily, have more sources and less pain in patients than BMSCs, which provid advantageous condition for allograft of ADSCs. Thus, increasing the undifferentiation ADSCs, inducing osteogenic differentiation and increasing bone formation may have preventing and treating effects with osteoporosis.
Glucocorticoid has been found to induce osteoporosis rats. Long-term and high-dose glucocorticoid could lead to abnormality of calcium and phosphorus metabolism, increase of parathormone, decrease osteoprotegerin. Proliferation and activity of osteoblasts could be inhibited while activity of osteoclast upgraded, which could induce bone resorption increasing and bone formation decreasing, lead to osteoporosis finally.
The aim of this experiment is to investigate the effect of bone mineral density, biomechanical properties and histomorphometry in glucocorticoid-induced osteoporosis (GIOP) by systemic transplantation of ADSCs, to explore a new method for prevention and treatment of osteoporosis in clinical.
Objective
1. To detach and culture the ADSCs from the adipose tissue of rats, and to test their multiple differentiation multipotent and surface markers. To judge that whether the ADSCs exist in the supporting-storing system;
2. To judge whether glucocorticoid-induced osteoporosis model succeed by detecting bone mineral density.
3. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone mineral metabolism and bone formation of GIOP rats by detecting the levels of serum Ca、P and ALP.
4. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone weight of GIOP rats by detecting bone mineral density.
5. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone strength and bone stiffness of GIOP rats by three-point bending test.
6. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on microenvironment in bone tissue of GIOP rats by histomorphometry.
7. To provide the evidence for the theory of "fasciology hypothesis", which consider that mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative diseases and aging diseases.8. Different bone filling materials were observed the morphology of bone in ovariectomized rats were filled, and provides some basis for the clinical application of new materials.
Methods
1. To detach the ADSCs from the rats'adipose tissue, and then cultured the cells in vitro. The6th passage cells were observed by their morphology, differentiatial potent by induced them into adipogenesis and osteogenesis, and their biological markers on the cell membrane, such as CD11b、CD29、CD45、CD49d、CD90and CD106. Then to judge whether the cultured cells were ADSCs.
2.40female Wistar rats were randomly divided into4groups (A, B, C and D),10in each group. Rats in B, C and D groups were injected with prednisolone into the neck subcutaneously at a dose of8mg/kg of body weight, three times per week for12weeks. The rats in group A were injected with physiological saline at the same volume for12weeks. Group A and B were sacrificed and specimens were procured to judge whether model succeed after12weeks.
3. Harvest and culture purified ADSCs from Wistar rats. After the GIOP models were succeed, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the6th passage were harvested and resuspended in the physiological saline at3×106cells/mL. Each rat in group C was injected with3×106of ADSCs into its tail vein. The rats in groups D were similarly administered with same volume of physiological saline. Sacrificed and drew the materials after4weeks.
4. After the rats were anesthetized by chloralization, the blood was got from the hearts of rats into the Pro-Coagulation Tubes, and stored them at room temperate for8h. Then centrifugated15min by the speed of3000r/min to prepare serum, and stored at-70℃as backup. The levels of serum Ca、P and ALP were measured respectively by azo Ⅲ PMA chromatometry and velocity method with automatic biochemistry analyzer. Then, Sacrificed the rats by heart bloodletting, took out of Third to Fifth lumbar vertebra and two sides of femur, and remove muscular tissue around. Bone densities at lumbar and right femur were measured by X-ray bone mineral density instrument with small animal measure software. Three-point bending tests were performed at the left femur using BOSE biomaterial dynamic test machine at a span of18mm and a displacement rate of0.05mm/s. Bones were positioned in the test apparatus with the dorsal side in compression and ventral side in tension. Mechanical properties measured included strength (ultimate force that the specimen sustained) and stiffness (the slope of the initial, linear portion of the load-deformation curve).
5. The up1/3parts of right tibias were got out after sacrificed, then split them across sagittal plane. The undecalcified slices were produced by used the methyl methacrylate, then stained by Giemsa and Von kossa, respectively. Used the light microscope to observe the histology and measure the histomorphometric parameters, included percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and osteoclast number (OC.N).
6. Osteoporosis model operation:normal female rats after successful anesthesia, abdominal shaving, iodophor disinfection. A ventral midline incision into the abdominal cavity, layer by layer. Find one ovary ligation and resection. Act with the removal of the other side. Suture of peritoneum and abdominal muscles, and skin suture. A week stitches.3months after the osteoporosis model.
7. The model is established successfully, make drilling wood vertical bone surface5mm deep a bone tunnel. In the tunnel inner implantation material and lightly compacted to the bone surface, then with bone wax sealing material implant operation:Zhou anteromedial shaving anesthesia after the success of double knee. Iodophor disinfection. From the knee anterior tibial spine incision along the length of about2cm. Step by step up into the periosteum, subperiosteal dissection revealed proximal tibia front inner side, with a diameter of3mm drill bit. Hemostatic, rinse, selected four kinds of biological materials of β-TCP,β-phosphate calcium:three comparison of CS calcium silicate:after degradation, the release of the beneficial element silicon,
Sr-CS strontium-doped calcium silicate:after degradation, release of the beneficial elements of silicon, strontium,45S5biochemical ceramic glass filled bone defect. Suture the wound (exposure). Animals were killed at9weeks after.
8. Application of HE staining, Giemsa, T-blue, Van Gieson, safranin O fast green staining. To observe the changes of bone slice morphology with different filling materials.
Results
1. Observation by light microscope, primary cultured ADSCs have the morphological features of long spindle, and polygon, and can form cell colony. The ADSCs were passaged at ratio1:3after7days, and then passaged every3days. Results In passage6th, the ADSCs have the homogenicity with a spindle or polygon shaped. The cells can be induced to differentiate into fat cells and osteoblasts. Flow cytometry demonstrated that CD29and CD90were high positive, CD106low positive, while CD11b、CD45and CD49d were negative.
2. Long-term and high-dose glucocorticoid can successfully establish osteoporosis rats model, which shows progressive decreasing of the level of serum Ca and ALP, as well as bone mineral density, bone strength and bone stiffness, but the levels of serum P increased obviously. Bone trabecular became thinner and disarranged, and destroyed with a wide intertrabecular distance, even had lacunae on the surface. The quantity, volume and thickness of bone trabecular were all decreased significantly, along with a significant increasing of trabecular separation and osteoclast number.
3. Transplanted allograft ADSCs could significantly increase the level of serum Ca and ALP, bone mineral density, bone strength and bone stiffness. The levels of serum P decreased obviously at the same time. Bone trabecular became thicken and harden with a increasing quantity. The quantity, volume and thickness of bone trabecular were all increased significantly but Trabecular separation and osteoclast number decreased.
Conclusion
1. There are ADSCs, one kind of undifferentiated mesenchymal stem cells, in supporting-storing system,
2. Rats injected with long-term and high-dose glucocorticoid can successfully establish osteoporosis model.
3. Through transplanting ADSCs into the osteoporosis rats can effectively improve Ca and P metabolism, increase bone formation, effectively prevent and treat osteoporosis, which provid a new method of preventing and treating GIOP in clinical.
4. This experiment provided the evidence for one theory of "fasciology hypothesis", mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative disease.
5. Different bone filling materials for bone trauma, surface effects. New materials of CS and Sr can promote bone formation.
In summary, transplanting ADSCs to glucocorticoid-induced osteoporosis rats have effects on bone formation, increasing bone density, improving biomechanics property, enhancing bone strength, thereby decrease incidence of bone fracture. The study further tell us that we should pay more attention to increase quantity of stem cells and induce them osteogenesis in research of GIOP, to provid a new research pathway of preventing and treating GIOP in clinical.
骨质疏松主要的特征是骨量减少和骨微结构破坏,导致骨脆性增加同时易于骨折的代谢性疾病。随着老龄化社会的发展,骨质疏松患者不断增多,如何有效预防和治疗骨质疏松已经成为国内外的热门话题。根据原林教授提出的“筋膜学说”：人体可以看做是由尚未分化的非特异性结缔组织(筋膜)支架构成的支持与储备系统与被该支架支持与包绕的已分化为各种功能细胞构成的功能系统共同组成。建立在各种功能细胞的专能特化的基础上的功能系统是被以筋膜支架中未分化的干细胞为核心的支持与储备系统支持和包绕的。支持与储备系统内未分化干细胞在微环境下通过分化为各种定向干细胞,源源不断的为功能系统中各种功能细胞的更新提供细胞源泉,同时为功能系统中的各种功能细胞的代谢提供营养,维持其内环境的稳定。因此,在支持与储备系统的支持下功能系统才可能维持结构和功能的稳定,当某种功能细胞在代谢过程中不能及时得到新细胞的补充,出现代谢障碍,机体就可能会出现相应的病变。骨质疏松的发病机制正是在骨组织吸收后,筋膜结缔组织支架中未分化的干细胞未能充分向成骨细胞分化,造成骨形成不足而出现的一种疾病。因此,如何干预骨代谢过程中出现的负平衡,如何有效保证并增加筋膜结缔组织支架中未分化的干细胞,使成骨细胞得到有效的补充,促进骨的形成,重建骨的正平衡是治疗骨质疏松的关键。
目前,骨缺损的修复仍是骨科临床中的棘手难题之一,以往研究显示采用骨移植治疗骨缺损的方法取得了一定的效果,但此方法存在较大的局限性。自体骨移植一直以来都被认为是骨缺损修复的一个金标准,由于植取骨量有限,已形成供区畸形,取骨手术也同样增加了患者的创伤痛苦；异体骨移植虽然取材方便,易获取,可减少手术次数,但是由于其具有抗原性,可引起机体的免疫排斥反应,同时存在着潜在传播疾病的风险,故异体骨移植的并发症也较多,如感染、骨折、骨不愈合等等。因此,人们对新型移植替代物的需求也不断增加。目前各种骨水泥已经备受青睐。伴随人口老龄化的发展,骨质疏松症已成为威胁着中老年人健康的主要疾病。骨质疏松极易并发骨折,同时这骨折也常伴有松质骨压缩及骨缺损。伴随着骨质疏松绝对人口的增加,因各种原因引起的骨缺损合并骨质疏松的患者也逐年增加。当机体骨质疏松时,由于破骨细胞活性增强,骨的吸收大于骨的形成,故新骨生成能力下降,导致骨质疏松骨缺损的愈合时间明显延迟,而新生骨痂减少,愈合后的骨生物力学强度差,容易骨折。与常规骨缺损的治疗相比,治疗骨质疏松骨缺损的花费要更大且传统方法疗效欠佳。骨水泥已逐渐被应用到骨质疏松骨折骨缺损的辅助治疗中,它一方而可增强固定螺钉,防止骨质疏松骨折内固定的松动,另一方面填充骨折的缺损,使其复位及愈合更好,减少发生骨折后遗症。磷酸钙骨水泥近几年也被应用于椎体成形术来治疗骨质疏松椎体压缩骨折,且疗效良好。缓释性骨长因子也渐渐被应用于骨质疏松骨缺损的治疗中,这显著加速了骨缺损的愈合,还在一定程度上改善局部的骨质疏松,增强局部骨生物力学,降低骨折的发生几率。
脂肪组织同样也是两系统理论中的支持与储备系统中重要组成部分,其分离出的脂肪源干细胞(adipose-derived stem cells, ADSCs)是筋膜结缔组织支架中未分化干细胞的一种重要储备形式。脂肪源干细胞的研究逐渐成为干细胞研究的热点。脂肪源干细胞易于获取,便于分离培养；具有多向分化潜能,能跨胚层分化为三胚层内的组织细胞,在特定的环境中可以向成骨、成软骨、成脂、成肌和成神经等诱导分化：具有免疫原性低和调节免疫功能。此外,骨髓间充质干细胞相比,脂肪源干细胞来源更广泛,获取更容易,对于病人而言,带来的创伤和痛苦更小。这些特征为脂肪源干细胞的同体及异体移植提供了更有利条件。因此,通过增加机体筋膜结缔组织内的未分化的脂肪源干细胞,促进其成骨分化,增加骨形成,可能会对骨质疏松起到预防和治疗作用。
利用糖皮质激素建立骨质疏松模型是目前国际上比较公认的骨质疏松症药物模型。长期注射大剂量的糖皮质激素可引起体内钙磷代谢的紊乱,促进甲状旁腺素的分泌,抑制骨保护素的分泌,抑制成骨细胞增殖和活性,增加破骨细胞数量和活性等多种途径促使骨形成减少,骨吸收增加,从而导致骨质疏松的发生。
为了探讨异体脂肪源干细胞移植及不同骨填充材料对骨质疏松的预防和治疗作用,本实验通过观察异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠体内血清钙及磷代谢、骨密度、骨生物力学及骨组织形态计量学等方面的影响进行综合评价,同时通过观察不同填充材料对去卵巢骨质疏松大鼠的骨切片的影响,探索一种新的骨质疏松治疗方法,为临床骨质疏松的预防和治疗提供新的研究思路。
目的
1、体外分离培养腹股沟皮下脂肪源干细胞,并对其进行多向分化能力和表面标志的鉴定,以判断在支持与储备系统中是否存在未分化的脂肪源干细胞；
2、通过对骨密度检测,判断长期给予糖皮质激素是否成功模拟建立骨质疏松模型?
3、通过检测大鼠钙、磷、碱性磷酸酶,观察异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠的骨体内钙、磷代谢和骨形成的影响；
4、通过检测实验大鼠的骨密度,观察异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠骨质量的影响；
5、通过检测三点弯曲试验,观察移植异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠的骨强度和骨刚度的影响；
6、通过对骨组织形态计量学观察,探索异体脂肪源干细胞移植对糖皮质激素致骨质疏松大鼠骨组织微环境的影响；
7、为进一步证实“筋膜学说”中“通过动员支持与储备系统中未分化干细胞可以治疗退行性和衰老性疾病”提供部分实验依据。
8、通过不同骨填充材料对去卵巢大鼠进行填充后进行相应骨形态学观察,筛选最佳骨材料,为临床新材料的应用提供部分依据。方法
1、取成年SD大鼠腹股沟皮下脂肪垫,I型胶原酶消化,体外分离培养筋膜结缔组织中脂肪源干细胞。分别通过形态学、成脂成骨诱导分化及利用流式细胞术对表面抗原CD11b、CD29、CD45、CD49d、CD90和CD106进行鉴定4代脂肪源干细胞,判断所培养的细胞是否为具有多向分化潜能的脂肪源干细胞。
2、40只SD雌性大鼠随机分成空白对照组(A组)、模型组(B组)、治疗组(C)和治疗对照组(D组)。B、C和D组分别给予颈背部皮下注射醋酸泼尼松龙注射液8mg/kg,3次/周；A组给予颈背部皮下注射生理盐水0.8mL,3次/周。12周后,将A组和B组处死取材,判断造模是否成功。
3、模拟骨质疏松模型成功后,收集传至第4代的脂肪源干细胞,经尾静脉向C组大鼠每只注射脂肪源干细胞3×106个,D组大鼠每只注射1mL生理盐水,4周后处死取材。
4、应用水合氯醛麻醉大鼠后,用促凝管接取心脏血液,室温下放置8h后3000r/min,离心15min后制备血清,分装后-70℃保存。全自动生化分析仪分别利用偶氮砷Ⅲ法、磷钼酸比色法及速率法测定血清中钙、磷和碱性磷酸酶的含量。取血完毕后,采用心脏放血处死,分别取出大鼠双侧股骨和第三至第五腰椎,去除周围肌肉组织。利用双能X线骨密度仪小动物测量软件测定腰椎和右侧股骨的骨密度值。利用BOSE生物材料动态力学试验机对左侧股骨进行三点弯曲实验,股骨背侧向上,腹侧向下,跨距为18mm,速率为0.05mm/s,股骨压断时记录最大载荷和刚度值。
5、处死大鼠后取出右侧胫骨上1/3部分,手术刀片矢状面剖开,固定后酒精逐级脱水,分别浸入浸液I(甲基丙烯酸甲酯70mL+邻苯二甲酸二丁酯30mL)、浸液Ⅱ(浸液I+过氧化苯甲酰1g)和浸液Ⅳ(浸液Ⅰ+过氧化苯甲酰2.5g)各2d,再用新鲜配制的浸液Ⅲ进行包埋。Jung K重型切骨机连续切取6μm厚度切片,分别进行组织学观察(Gemisa染色)和类骨质观察(von Kossa染色)。测量区域为生长板下1mm至4mm范围内,采用Lecia QWin多功能彩色病理图像分析软件进行骨组织形态计量学分析。具体参数包括骨小梁相对体积(BV/TV)X骨小梁厚度(Tb.Th)、骨小梁数量(Tb.N)和骨小梁分离度(Tb.Sp)和单位骨小梁破骨细胞数(OC.N),均采用国际通用的公式进行计算。
6、骨质疏松模型手术：正常雌性大鼠全麻成功后,腹部剃毛,碘伏消毒。下腹正中切口,逐层进入腹腔。找出一侧卵巢结扎切除。同法切除另一侧。缝合腹膜及腹肌,最后缝合皮肤。一周拆线。3个月后即为骨质疏松模型。
7、待模型建立成功后,做材垂直骨面钻孔深5毫米建立骨隧道。于隧道内植入材料并轻轻压实至骨面,然后用骨蜡封堵料植入手术：全麻成功后双下肢膝周前内侧剃毛。碘伏消毒。从膝部沿胫骨前棘切口长约2cm。逐层进达骨膜下,骨膜下剥离显露胫骨近端前内侧面,用直径3毫米钻头。止血,冲洗,选取四种生物材料： β-磷酸三钙(p-TCP);硅酸钙(CS),降解后,释放有益元素硅；掺锶硅酸钙(Sr-CS),降解后,释放有益元素硅、锶；45s5生化陶瓷玻璃进行填充骨缺损。缝合伤口。9周后处死取材。
8、应用HE染色、Giemsa、T-blue、Van gieson、番红O&固绿进行染色。观察不同填充材料骨切片形态学变化。
结果
1、经光学显微镜观察,原代培养的ADSCs在24h后大部分可贴壁,细胞呈梭形、圆形、多边形等。细胞长势良好,7d后可以1：3比例传代,之后每3d传一次代。经换液传代至第4代,可见细胞形态均一,呈纺锤形或者多角形。体外可以向成脂、成骨诱导分化。流式细胞术检测发现CD29和CD90呈强阳性,CD106呈弱阳性,CD11b、CD45和CD49d呈阴性。
2、长期大剂量给予糖皮质激素后,与空白对照组大鼠血清钙(3.63±0.20)、血清磷水平(3.12±0.83)和碱性磷酸酶水平(203.8±40.36)、腰椎骨密度(162.70±14.77)、股骨骨密度(254.40±28.95)、骨强度(98.22±10.72)和骨刚度(192.66±42.28)比较,模型组大鼠血清钙(2.60±0.18)和碱性磷酸酶水平(149.50±22.21)、腰椎骨密度(115.70±16.87)、股骨骨密度(183.40±12.28)、骨强度(58.88±12.53)和骨刚度(127.53±19.13)均显著降低,而血清磷水平(5.25±1.11)显著上升(P<0.05)；骨小梁变细、变薄,骨小梁排列紊乱,骨小梁间出现断裂,间距增大,出现陷窝；骨小梁的数量(4.95±0.75)、相对体积(17.31±3.66)和厚度(35.82±3.88)均显著降低(P<0.05),而骨小梁的分离度(171.30±29.72)增高,单位骨小梁破骨细胞数(52.70±10.34)增多(P<0.05)。
3、异体移植脂肪源干细胞后,与模型组比较,治疗组大鼠血清钙(3.01±0.30)和碱性磷酸酶水平(202.5±40.82)、股骨骨密度(226.50±25.54)、(腰椎骨密度149.30±12.52)、骨强度(80.37±9.37)和骨刚度(187.10±30.06)均显著增加,而血清磷水平(2.84±1.78)显著降低(P<0.05)；骨小梁数目增多,骨小梁变厚变粗,骨小梁间出现连接,陷窝减少；骨小梁的数量(6.42±1.24)、相当体积(25.00±4.88)和厚度(39.63±4.16)显著增加,而骨小梁分离度(124.72±29.79)显著降低,单位骨小梁破骨细胞数(29.52±8.38)减少(P<0.05)。
4、各种染色结果显示p-磷酸三钙组9w无炎症反应；而CS硅酸钙和Sr-CS掺锶硅酸钙：相对TCP有较多的成骨,但9w仍有炎症反应。结论
1、两系统理论中的支持与储备系统中含有脂肪源干细胞,并且这种干细胞是一种未分化的间充质干细胞；
2、长期给予大剂量的糖皮质激素可以模拟骨质疏松模型；
3、通过移植异体脂肪源干细胞可有效改善体内钙、磷代谢,增加骨形成,有效预防和治疗骨质疏松,为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法；
4、对“筋膜学说”中“通过动员筋膜结缔组织中储备的未分化干细胞可治疗退行性疾病”提供了部分实验依据。
5、不同的骨填充材料,对骨创伤面影响不同。新材料CS和Sr均可促进骨形成。为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法。
综上所述,移植异体脂肪源干细胞对糖皮质激素致骨质疏松大鼠有一定的治疗及预防作用,能够促进骨形成,增加骨密度,改善骨生物力学性能,增强骨强度,降低骨折的发生率。本实验进一步提示在骨质疏松的研究中应该更加关注提高机体干细胞的数量和促进干细胞向成骨细胞分化及骨组织的形成方面,为临床上治疗骨质疏松提供了一种新的研究思路。同时,不同填充骨质疏松骨缺损的新材料填充后骨切片对比,新材料CS和Sr均可促进骨形成。为临床上骨质疏松的研究和治疗提供了一种新的研究思路和方法。
Background:
Osteoporosis is a metabolic disease that characteristic by osteopenia and bone microstructural destroy, which could cause fragility increasing and able to fractured. Due to the increasing osteoporosis patients, research that prevent and treat osteoporosis have an important clinical significance. Recently, Prof. L. Yuan had put forward a new hypothesis, fasciology hypothesis. In fasciology, the human body is classified into two major systems. One is the supporting-storing system, which is consisted of is constituted of undifferentiated cells from the network of unspecialized connective tissues (fascia network). The other one is the functional system, which is consisted of differentiated functional cells and is enclosed by the supporting-storing system. The undifferentiated stem cells in the supporting-storing system incessantly differentiate into functional cells. Undifferentiated stem cells in the supporting-storing system incessantly migrate to target areas, differentiate into committed-stem cells, and further differentiate into functional cells. The supporting-storing system provides energy and cell reserve for the functional system. The structures and functions of an organism are maintained by the incessant supplement and refreshment from the supporting-storing system to the functional system. Meanwhile, under the regulation of the nervous system and immune system, the fascia network throughout the body regulates the functional and living status of cells and provides a stable environment for the survival of functional cells.
The repair of bone defects is one of the common difficult problem in clinical department of orthopedics, the bone transplantation in the treatment of bone defect has obtained the certain effect, but this method has limitation. Autogenous bone transplantation has been considered the gold standard for the repair of bone defect, but the bone graft is limited, can cause donor deformity, bone operation also additional trauma and anguish; allogeneic bone transplantation can reduce the times of operation, convenient, but because of its antigenicity, can cause immune rejection at the same time, there is the potential spread of disease risk, and allograft bone complications are more, such as infection, fracture and nonunion. Therefore, the demand for the new substitute transplantation increasing. Bone cement has favored. With the increasing of aging population, osteoporosis has become a major disease that threatens the health of middle-aged and elderly people, osteoporosis is very easy to cause fracture, the fractures are often accompanied with cancellous bone compression and bone defects, in addition with the increase of osteoporosis absolute population, due to various causes of patients with bone defect with osteoporosis caused by more and more. Under circumstance of osteoporosis, enhance the activity of osteoclasts, bone resorption and bone formation, decrease new bone formation ability, bone defect healing was delayed, the new callus biomechanical strength reduction, bone healing after the poor fracture easily, therefore, compared with the conventional bone defect, bone defect costly and traditional methods poor efficacy. Auxiliary treatment of bone cement is gradually applied in osteoporotic fracture bone defect, it can enhance the fixing screw pull-out strength, prevent osteoporosis fracture internal fixation loosening, filling the bone defect, make better fracture reduction and healing, reduce the occurrence of sequelae of fracture, in recent years, calcium phosphate cement was used in vertebroplasty surgery for the treatment of osteoporotic vertebral compression fractures, and achieved good effect. Treatment of sustained-release bone length factor has gradually been applied in osteoporotic bone defects, which not only significantly accelerated the healing of bone defects, but also can improve the local osteoporosis in a certain extent, enhancing local bone biomechanics, to reduce the incidence of fracture.
According to fasciology, the process of a life is just that of continuous consumption of the supporting-storing system. The supplementary of functional cells to repair the functions of damaged tissues ensures normal activities of the functional system. Once one of the functional cells could not be updated by the supporting-storing system in metabolism, the body will be disorder. Osteoporosis is the disease that the osteoblasts, supplemented from the undifferentiated stem cells in the supporting-storing system, is insufficient after bone resorption caused by many factors, e.g. menopause, age and glucocorticoid et al. Therefore, when negative balance exists in bone metabolism, how to increase the undifferentiated stem cells in the supporting-storing system to differentiate into osteoblasts may be an effective treatment for osteoporosis.
Adipose-derived stem cells (ADSCs), which isolated from adipose tissue, are main undifferentiated cells in the supporting-storing system. Currently, ADSCs have become one of the focuses in stem cell research. They are easy to be cultured, rich in source and have differentiation of multipotent, even low in immunity. They also have the capacity of cross-mesodermal differentiation, such as osteogenic, chondrogenic, adipogenic, and myogenic et al. Furthermore, ADSCs are isolated more easily, have more sources and less pain in patients than BMSCs, which provid advantageous condition for allograft of ADSCs. Thus, increasing the undifferentiation ADSCs, inducing osteogenic differentiation and increasing bone formation may have preventing and treating effects with osteoporosis.
Glucocorticoid has been found to induce osteoporosis rats. Long-term and high-dose glucocorticoid could lead to abnormality of calcium and phosphorus metabolism, increase of parathormone, decrease osteoprotegerin. Proliferation and activity of osteoblasts could be inhibited while activity of osteoclast upgraded, which could induce bone resorption increasing and bone formation decreasing, lead to osteoporosis finally.
The aim of this experiment is to investigate the effect of bone mineral density, biomechanical properties and histomorphometry in glucocorticoid-induced osteoporosis (GIOP) by systemic transplantation of ADSCs, to explore a new method for prevention and treatment of osteoporosis in clinical.
Objective
1. To detach and culture the ADSCs from the adipose tissue of rats, and to test their multiple differentiation multipotent and surface markers. To judge that whether the ADSCs exist in the supporting-storing system;
2. To judge whether glucocorticoid-induced osteoporosis model succeed by detecting bone mineral density.
3. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone mineral metabolism and bone formation of GIOP rats by detecting the levels of serum Ca、P and ALP.
4. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone weight of GIOP rats by detecting bone mineral density.
5. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on bone strength and bone stiffness of GIOP rats by three-point bending test.
6. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on microenvironment in bone tissue of GIOP rats by histomorphometry.
7. To provide the evidence for the theory of "fasciology hypothesis", which consider that mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative diseases and aging diseases.8. Different bone filling materials were observed the morphology of bone in ovariectomized rats were filled, and provides some basis for the clinical application of new materials.
Methods
1. To detach the ADSCs from the rats'adipose tissue, and then cultured the cells in vitro. The6th passage cells were observed by their morphology, differentiatial potent by induced them into adipogenesis and osteogenesis, and their biological markers on the cell membrane, such as CD11b、CD29、CD45、CD49d、CD90and CD106. Then to judge whether the cultured cells were ADSCs.
2.40female Wistar rats were randomly divided into4groups (A, B, C and D),10in each group. Rats in B, C and D groups were injected with prednisolone into the neck subcutaneously at a dose of8mg/kg of body weight, three times per week for12weeks. The rats in group A were injected with physiological saline at the same volume for12weeks. Group A and B were sacrificed and specimens were procured to judge whether model succeed after12weeks.
3. Harvest and culture purified ADSCs from Wistar rats. After the GIOP models were succeed, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the6th passage were harvested and resuspended in the physiological saline at3×106cells/mL. Each rat in group C was injected with3×106of ADSCs into its tail vein. The rats in groups D were similarly administered with same volume of physiological saline. Sacrificed and drew the materials after4weeks.
4. After the rats were anesthetized by chloralization, the blood was got from the hearts of rats into the Pro-Coagulation Tubes, and stored them at room temperate for8h. Then centrifugated15min by the speed of3000r/min to prepare serum, and stored at-70℃as backup. The levels of serum Ca、P and ALP were measured respectively by azo Ⅲ PMA chromatometry and velocity method with automatic biochemistry analyzer. Then, Sacrificed the rats by heart bloodletting, took out of Third to Fifth lumbar vertebra and two sides of femur, and remove muscular tissue around. Bone densities at lumbar and right femur were measured by X-ray bone mineral density instrument with small animal measure software. Three-point bending tests were performed at the left femur using BOSE biomaterial dynamic test machine at a span of18mm and a displacement rate of0.05mm/s. Bones were positioned in the test apparatus with the dorsal side in compression and ventral side in tension. Mechanical properties measured included strength (ultimate force that the specimen sustained) and stiffness (the slope of the initial, linear portion of the load-deformation curve).
5. The up1/3parts of right tibias were got out after sacrificed, then split them across sagittal plane. The undecalcified slices were produced by used the methyl methacrylate, then stained by Giemsa and Von kossa, respectively. Used the light microscope to observe the histology and measure the histomorphometric parameters, included percent trabecular area (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and osteoclast number (OC.N).
6. Osteoporosis model operation:normal female rats after successful anesthesia, abdominal shaving, iodophor disinfection. A ventral midline incision into the abdominal cavity, layer by layer. Find one ovary ligation and resection. Act with the removal of the other side. Suture of peritoneum and abdominal muscles, and skin suture. A week stitches.3months after the osteoporosis model.
7. The model is established successfully, make drilling wood vertical bone surface5mm deep a bone tunnel. In the tunnel inner implantation material and lightly compacted to the bone surface, then with bone wax sealing material implant operation:Zhou anteromedial shaving anesthesia after the success of double knee. Iodophor disinfection. From the knee anterior tibial spine incision along the length of about2cm. Step by step up into the periosteum, subperiosteal dissection revealed proximal tibia front inner side, with a diameter of3mm drill bit. Hemostatic, rinse, selected four kinds of biological materials of β-TCP,β-phosphate calcium:three comparison of CS calcium silicate:after degradation, the release of the beneficial element silicon,
Sr-CS strontium-doped calcium silicate:after degradation, release of the beneficial elements of silicon, strontium,45S5biochemical ceramic glass filled bone defect. Suture the wound (exposure). Animals were killed at9weeks after.
8. Application of HE staining, Giemsa, T-blue, Van Gieson, safranin O fast green staining. To observe the changes of bone slice morphology with different filling materials.
Results
1. Observation by light microscope, primary cultured ADSCs have the morphological features of long spindle, and polygon, and can form cell colony. The ADSCs were passaged at ratio1:3after7days, and then passaged every3days. Results In passage6th, the ADSCs have the homogenicity with a spindle or polygon shaped. The cells can be induced to differentiate into fat cells and osteoblasts. Flow cytometry demonstrated that CD29and CD90were high positive, CD106low positive, while CD11b、CD45and CD49d were negative.
2. Long-term and high-dose glucocorticoid can successfully establish osteoporosis rats model, which shows progressive decreasing of the level of serum Ca and ALP, as well as bone mineral density, bone strength and bone stiffness, but the levels of serum P increased obviously. Bone trabecular became thinner and disarranged, and destroyed with a wide intertrabecular distance, even had lacunae on the surface. The quantity, volume and thickness of bone trabecular were all decreased significantly, along with a significant increasing of trabecular separation and osteoclast number.
3. Transplanted allograft ADSCs could significantly increase the level of serum Ca and ALP, bone mineral density, bone strength and bone stiffness. The levels of serum P decreased obviously at the same time. Bone trabecular became thicken and harden with a increasing quantity. The quantity, volume and thickness of bone trabecular were all increased significantly but Trabecular separation and osteoclast number decreased.
Conclusion
1. There are ADSCs, one kind of undifferentiated mesenchymal stem cells, in supporting-storing system,
2. Rats injected with long-term and high-dose glucocorticoid can successfully establish osteoporosis model.
3. Through transplanting ADSCs into the osteoporosis rats can effectively improve Ca and P metabolism, increase bone formation, effectively prevent and treat osteoporosis, which provid a new method of preventing and treating GIOP in clinical.
4. This experiment provided the evidence for one theory of "fasciology hypothesis", mobilizating undifferentiated stem cell of supporting-storing system could treat degenerative disease.
5. Different bone filling materials for bone trauma, surface effects. New materials of CS and Sr can promote bone formation.
In summary, transplanting ADSCs to glucocorticoid-induced osteoporosis rats have effects on bone formation, increasing bone density, improving biomechanics property, enhancing bone strength, thereby decrease incidence of bone fracture. The study further tell us that we should pay more attention to increase quantity of stem cells and induce them osteogenesis in research of GIOP, to provid a new research pathway of preventing and treating GIOP in clinical.
引文
[1]贾经汉,邱新建,陈志坚.常用骨质疏松动物模型特点的综述[J].广西中医学院学报.2006,9(4)：76-79.
[2]贾经汉,邱新建,陈志坚.骨质疏松动物模型的研究进展[J].中国康复医学杂志.2007,22(8)：765-768.
[3]尚庆,刘宗超,鲁晓波.骨质疏松动物模型特点的综述[J].中国实用医药.2008,3(13)：193-195.
[4]何冬丽,米淑筠.骨质疏松症治疗及预防进展(综述)[J].天津护理.2001,9(5)：259-261.
[5]崔双,薛昊罡.绝经后女性骨质疏松患者骨密度与血脂和护骨素的相关性[J].中国老年学杂志.2012,32(10)：2051-2053.
[6]于文广,张鲁阳,张学顺.类风湿关节炎患者骨质疏松健康教育相关因素调查分析[J].中国骨质疏松杂志.2012,18(7)：623-627.
[7]邓娟,裴必伟,徐胜前,等.类风湿关节炎患者全身骨质疏松和局部骨侵蚀的关系研究[J].中华风湿病学杂志.2012,16(10)：674-678.
[8]唐鹏,孟国林,王军,等.长期应用双膦酸盐类药物对老年性骨质疏松骨折愈合过程的影响[J].中国骨质疏松杂志.2012,18(4)：317-322.
[9]刘刚,唐瑭,刘晶晶.阿仑膦酸钠联合钙尔奇D治疗老年骨质疏松62例[J].中国老年学杂志.2012,32(18)：4070-4071.
[10]高天君,杨达宇.比较切开复位内固定和石膏固定治疗老年骨质疏松性桡骨远端骨折对患肢继发性骨质疏松和关节功能的影响[J].中国骨质疏松杂志.2012,18(3)：245-247.
[11]郭庆华.老年性骨质疏松症药物治疗的新进展(综述)[J].中国城乡企业卫生.2004(4)：25-26.
[12]邓海鸥,李东风,张伟杰,等.静脉用唑来膦酸治疗老年骨质疏松患者时引起的急性期发热反应分析[J].南方医科大学学报.2011,31(12)：2076-2078.
[13]汪元浚,杨发满,李蓉,等.综合营养干预措施对老年原发性骨质疏松患者骨密度的影响[J].中国老年学杂志.2012,32(4)：727-728.
[14]高俊,张前德,张曦,等.废用性骨质疏松动物模型的建立与评价[J].广东医学.2012,33(18)：2728-2731.
[15]廖立,杨小红,金岩.骨质疏松发病过程中骨髓间充质干细胞差异性表达microRNA的筛选及其在干细胞多向分化中的功能[J].浙江大学学报(医学版).2012,41(1)：75-80.
[16]袁绍辉,刘伟,吴滨奇,等.骨质疏松性骨折愈合中骨改建的动态参数及骨密度变化[J].中国组织工程研究与临床康复.2011,15(41)：7627-7630.
[17]孙允高,罗先正,阮祥燕,等.骨质疏松诊断技术的新进展[J].仪器仪表学报.2000,21(Z1)：25-27,40.
[18]钱慕周,尹友生,何勇,等.雷洛昔芬对血液透析妇女骨质疏松的干预作用[J].山东医药.2012,52(12)：83-85.
[19]项丽娟,张艳,庞炜.运动干预骨质疏松大鼠对二甲基精氨酸二甲胺水解酶表达的影响[J].山东体育学院学报.2011,27(8)：62-65.
[20]成强.优选组合用药治疗老年骨质疏松的疗效[J].中国老年学杂志.2012,32(15)：3318-3320.
[21]单光宇,薛昊罡.糖尿病骨质疏松大鼠AGEs的表达及其与TGF-p1的相关性[J].中国老年学杂志.2012,32(11)：2344-2345.
[22]任炼,李志忠,隋杰,等.骨水泥充填椎体成形治疗骨质疏松性椎体压缩骨折：6年随访[J].中国组织工程研究.2012,16(12)：2110-2114.
[23]杨宁,崔岳毅,韩晓光,等.骨质疏松大鼠股骨内单次注射辛伐他汀强化骨骼的实验研究[J].中华骨科杂志.2012,32(5)：500-506.
[24]范光磊,薄静莉,陈杰敏,等.骨转换生化标志物与老年2型糖尿病患者并发骨质疏松的相关性研究[J].中华老年医学杂志.2012,31(8)：657-661.
[25]杨希重,季翠杰,王德春,等.去卵巢骨质疏松模型大鼠的番茄红素干预[J].中国组织工程研究.2012,16(15)：2750-2756.
[26]刘少军,张娴,冯丽敏,等.应力缺失致骨质疏松大鼠成骨细胞细胞信号外调节激酶表达及通络生骨胶囊的影响[J].中国组织工程研究与临床康复.2011,15(20)：3625-3628.
[1]Migliaccio S, Brama M, Malavolta N. Management of glucocorticoids-induced osteoporosis: role of teriparatide[J]. Ther Clin Risk Manag.2009,5(2):305-310.
[2]Adler R A. Glucocorticoid-induced osteoporosis:management update[J]. Curr Osteoporos Rep.2010,8(1):10-14.
[3]王迎军,杜昶,赵娜如,等.仿生人工骨修复材料研究[J].华南理工大学学报(自然科学版).2012,40(10)：51-58.
[4]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[5]许宋锋,胡蕴玉,林开利,等.多孔硅酸钙生物陶瓷体内非骨性环境的植入研究[J].无机材料学报.2008,23(3)：611-616.
[6]刘冰,李淑娟,杨冬茹,等.大黄素治疗实验性牙周炎的骨计量学研究[J].实用口腔医学杂志.2010,26(5)：593-596.
[7]Parfitt A M, Drezner M K, Glorieux F H, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res.1987,2(6): 595-610.
[8]王学英.骨质疏松症防治药物发展现状[J].青海医药杂志.2008,38(11)：75-77.
[9]尚庆,刘宗超,鲁晓波.骨质疏松动物模型特点的综述[J].中国实用医药.2008,3(13)：193-195.
[10]陈璐,周文清,吴燕平,等.p-磷酸三钙复合自体骨移植在上颌窦闭合式提 升中的应用[J].上海口腔医学.2011,20(3)：282-285.
[11]姚志鹏,刘文革,倪国新.掺锶羟基磷灰石骨水泥的生物学特点及在骨科的应用[J].中国组织工程研究与临床康复.2008,12(36)：7151-7154.
[12]Kulak C A, Dempster D W. Bone histomorphometry:a concise review for endocrinologists and clinicians[J]. Arq Bras Endocrinol Metabol.2010,54(2): 87-98.
[13]Chavassieux P M, Arlot M E, Reda C, et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis[J]. J Clin Invest.1997,100(6):1475-1480.
[14]Thompson D D, Simmons H A, Pirie C M, et al. FDA Guidelines and animal models for osteoporosis[J]. Bone.1995,17(4 Suppl):125S-133S.
[15]Parfitt A M. Renal bone disease: a new conceptual framework for the interpretation of bone histomorphometry[J]. Curr Opin Nephrol Hypertens. 2003,12(4):387-403.
[16]裴玉岩,刘继光,李凌志,等.淫羊藿对骨质疏松家兔颌骨骨缺损愈合的影响[J].黑龙江医药科学.2011,34(1)：93-94.
[17]王世惟,裴庆国,宋萌.骨髓基质干细胞与β-TCP生物相容性的实验研究[J].上海口腔医学.2010,19(3)：319-322.
[18]沈宇辉,邓廉夫,潘浩波,等.含锶可降解生物陶瓷及生物玻璃骨植入材料研究进展[J].国际骨科学杂志.2011,32(2)：75-77.
[19]李峰,赵信义,吴军正,等.含锶磷酸钙骨水泥生物相容性评价[J].牙体牙髓牙周病学杂志.2006,16(5)：264-268.
[20]朱磊.微量元素在骨质疏松防治中的作用[J].上海医药.2012,33(8)：43-46.
[21]Lee G H, Hwang J D, Choi J Y, et al. An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts:the involvement of BAX inhibitor-1[J]. Int J Biochem Cell Biol.2011,43(9):1305-1317.
[22]袁月,哈斯达来,张劲松,等.碳化硅对成骨细胞增殖和分化的影响[J].生物医学工程与临床.2012,16(2)：109-113.
[1]Trzaska K A, Castillo M D, Rameshwar P. Adult mesenchymal stem cells in neural regeneration and repair: Current advances and future prospects (Review)[J]. Mol Med Report.2008,1(3):307-316.
[2]Augello A, Kurth T B, De Bari C. Mesenchymal stem cells:a perspective from in vitro cultures to in vivo migration and niches[J]. Eur Cell Mater.2010,20: 121-133.
[3]Kogler G, Sensken S, Wernet P. Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood[J]. Exp Hematol.2006,34(11):1589-1595.
[4]Fumimoto Y, Matsuyama A, Komoda H, et al. Creation of a rich subcutaneous vascular network with implanted adipose tissue-derived stromal cells and adipose tissue enhances subcutaneous grafting of islets in diabetic mice[J]. Tissue Eng Part C Methods.2009,15(3):437-444.
[5]Xiao Y, Mareddy S, Crawford R. Clonal characterization of bone marrow derived stem cells and their application for bone regeneration[J]. Int J Oral Sci. 2010,2(3):127-135.
[6]孙慧如,王英,董明敏.豚鼠脂肪干细胞的分离培养与鉴定[J].中国组织工程研究与临床康复.2007,11(7)：1213-1215.
[7]Han Z B, Chen H X, Deng J X. [Multipotential differentiation and potential applications of adipose-derived stem cells][J]. Sheng Wu Gong Cheng Xue Bao.2007,23(2):195-200.
[8]韩长杰,杨向群,张传森.脂肪干细胞的免疫学性质[J].中国组织工程研究与临床康复.2010,14(27)：5095-5098.
[9]Nakao N, Nakayama T, Yahata T, et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal stem cells[J]. Am J Pathol.2010,177(2): 547-554.
[10]Mosna F, Sensebe L, Krampera M. Human bone marrow and adipose tissue mesenchymal stem cells:a user's guide[J]. Stem Cells Dev.2010,19(10): 1449-1470.
[11]Casteilla L, Planat-Benard V, Bourin P, et al. [Use of adipose tissue in regenerative medicine.][J]. Transfus Clin Biol.2011.
[12]de Mattos C A, Alves A L, Golim M A, et al. Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue.[J]. Vet Immunol Immunopathol.2009,132(2-4):303-306.
[13]Sung J H, Yang H M, Park J B, et al. Isolation and characterization of mouse mesenchymal stem cells.[J]. Transplant Proc.2008,40(8):2649-2654.
[14]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional Chinese medicine[J]. J Acupunct Meridian Stud. 2010,3(2):125-132.
[15]原林,钟世镇.人体自体检测与调控系统(筋膜学)--经络有关的解剖学基础[J].天津中医药.2004,21(5)：356-359.
[16]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[17]Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies[J]. Tissue Eng.2001,7(2): 211-228.
[18]Choi Y S, Cha S M, Lee Y Y, et al. Adipogenic differentiation of adipose tissue derived adult stem cells in nude mouse[J]. Biochem Biophys Res Commun.2006,345(2):631-637.
[19]Luzi E, Marini F, Sala S C, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor[J]. J Bone Miner Res.2008,23(2):287-295.
[20]Gregory C A, Gunn W G, Peister A, et al. An Alizarin red-based assay of mineralization by adherent cells in culture:comparison with cetylpyridinium chloride extraction[J]. Anal Biochem.2004,329(1):77-84.
[21]Katz A J, Tholpady A, Tholpady S S, et al. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells[J]. Stem Cells.2005,23(3):412-423.
[22]Wan D C, Shi Y Y, Nacamuli R P, et al. Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling[J]. Proc Natl Acad Sci U S A. 2006,103(33):12335-12340.
[23]Madonna R, De Caterina R. Adipose tissue: a new source for cardiovascular repair[J]. J Cardiovasc Med (Hagerstown).2010,11(2):71-80.
[24]Yanez R, Lamana M L, Garcia-Castro J, et al. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease[J]. Stem Cells.2006,24(11): 2582-2591.
[25]Vieira N M, Brandalise V, Zucconi E, et al. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells[J]. Cell Transplant.2010,19(3):279-289.
[26]Mcintosh K, Zvonic S, Garrett S, et al. The immunogenicity of human adipose-derived cells:temporal changes in vitro[J]. Stem Cells.2006,24(5): 1246-1253.
[27]Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC[J]. Cell Biochem Funct.2008,26(6):664-675.
[28]Rigotti G, Marchi A, Sbarbati A. Adipose-derived mesenchymal stem cells: past, present, and future[J]. Aesthetic Plast Surg.2009,33(3):271-273.
[29]Ren Y, Wu H, Ma Y, et al. [Isolation, cultivation and identification of adipose-derived stem cell in bovines][J]. Sheng Wu Gong Cheng Xue Bao. 2010,26(12):1645-1651.
[30]Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential[J]. Cytotherapy.2003,5(5): 362-369.
[31]Schaffler A, Buchler C. Concise review:adipose tissue-derived stromal cells--basic and clinical implications for novel cell-based therapies.[J]. Stem Cells.2007,25(4):818-827.
[32]王栋,鹿均先,熊传芝.人脂肪干细胞分离培养鉴定及向成骨细胞诱导分化的实验研究[J].现代医学.2008,36(3)：190-193.
[33]Korn B S, Kikkawa D O, Hicok K C. Identification and characterization of adult stem cells from human orbital adipose tissue[J]. Ophthal Plast Reconstr Surg.2009,25(1):27-32.
[34]Gu F, Gao J H, Lu F. [Culture of the single cell clones of human adipose-derived stem cells and identification of their surface antigen expressions][J]. Nan Fang Yi Ke Da Xue Xue Bao.2008,28(6):1067-1069.
[35]Zola H, Swart B, Boumsell L, et al. Human Leucocyte Differentiation Antigen nomenclature:update on CD nomenclature. Report of IUIS/WHO Subcommittee.[J]. J Immunol Methods.2003,275(1-2):1-8.
[36]CD antigens 1996:updated nomenclature for clusters of differentiation on human cells. IUIS/WHO Subcommittee on CD Nomenclature.[J]. Bull World Health Organ.1997,75(4):385-387.
[37]Fukumori T, Takenaka Y, Yoshii T, et al. CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis.[J]. Cancer Res.2003,63(23): 8302-8311.
[38]Munakata Y, Saito T, Watanabe T, et al. Rapid inhibitory effect of tacrolimus on T cell migration by suppressing CD29-related functions.[J]. Clin Exp Rheumatol.2004,22(2):197-204.
[39]Rizzatti E G, Garcia A B, Portieres F L, et al. Expression of CD117 and CDllb in bone marrow can differentiate acute promyelocytic leukemia from recovering benign myeloid proliferation.[J]. Am J Clin Pathol.2002,118(1): 31-37.
[40]Paulli M, Rosso R, Kindl S, et al. Immunophenotypic characterization of the cell infiltrate in five cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease).[J]. Hum Pathol.1992,23(6): 647-654.
[41]Shaw E B. Visibility amplification of intestinal intraepithelial lymphocytes.[J]. Arch Pathol Lab Med.2002,126(8):897.
[42]Krasinskas A M, Wasik M A, Kamoun M, et al. The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation.[J]. Am J Clin Pathol. 1998,110(6):797-805.
[43]Chesney J, Metz C, Stavitsky A B, et al. Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.[J]. J Immunol.1998,160(1):419-425.
[44]Alon R, Feigelson S W, Manevich E, et al. Alpha4betal-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.[J]. J Cell Biol.2005,171(6):1073-1084.
[45]Soilu-Hanninen M, Laaksonen M, Hanninen A. Hyaluronate receptor (CD44) and integrin alpha4 (CD49d) are up-regulated on T cells during MS relapses.[J]. J Neuroimmunol.2005,166(1-2):189-192.
[46]Miller D H, Khan O A, Sheremata W A, et al. A controlled trial of natalizumab for relapsing multiple sclerosis.[J]. N Engl J Med.2003,348(1):15-23.
[47]Berger J R, Houff S. Progressive multifocal leukoencephalopathy:lessons from AIDS and natalizumab.[J]. Neurol Res.2006,28(3):299-305.
[48]Negrin R S, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34+Thy-1+hematopoietic stem cells in patients with metastatic breast cancer.[J]. Biol Blood Marrow Transplant.2000,6(3):262-271.
[49]Wetzel A, Chavakis T, Preissner K T, et al. Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD1 lb/CD18).[J]. J Immunol.2004,172(6):3850-3859.
[50]Nakamura Y, Muguruma Y, Yahata T, et al. Expression of CD90 on keratinocyte stem/progenitor cells.[J]. Br J Dermatol.2006,154(6):1062-1070.
[51]Chang A, Benda P M, Wood B L, et al. Lineage-specific identification of nonhematopoietic neoplasms by flow cytometry.[J]. Am J Clin Pathol.2003, 119(5):643-655.
[52]Johnson L A, Clasper S, Holt A P, et al. An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium.[J]. J Exp Med.2006,203(12):2763-2777.
[53]Cybulsky M I, Iiyama K, Li H, et al. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.[J]. J Clin Invest.2001,107(10):1255-1262.
[54]Barrier B F, Sharpe-Timms K L. Expression of soluble adhesion molecules in sera of women with stage III and IV endometriosis.[J]. J Soc Gynecol Investig. 2002,9(2):98-101.
[55]Cardier J E, Rivas B, Romano E, et al. Evidence of vascular damage in dengue disease:demonstration of high levels of soluble cell adhesion molecules and circulating endothelial cells.[J]. Endothelium.2006,13(5):335-340.
[56]Silva H C, Garcao F, Coutinho E C, et al. Soluble VCAM-1 and E-selectin in breast cancer: relationship with staging and with the detection of circulating cancer cells.[J]. Neoplasma.2006,53(6):538-543.
[57]Kim U, Shin D G, Park J S, et al. Homing of adipose-derived stem cells to radiofrequency catheter ablated canine atrium and differentiation into cardiomyocyte-like cells.[J]. Int J Cardiol.2009.
[58]Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells.[J]. Biochem Biophys Res Commun.2002,294(2):371-379.
[59]雷磊,廖威明,盛璞义,等.人类脂肪来源成体干细胞体外培养的生物学特征研究及供体年龄对其增殖的影响[J].中国科学(C辑：生命科学).2007(02).
[60]Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.[J]. Exp Hematol.2005,33(11):1402-1416.
[61]De U D, Alfonso Z, Zuk P A, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.[J]. Immunol Lett.2003,89(2-3):267-270.
[62]Mesimaki K, Lindroos B, Tornwall J, et al. Novel maxillary reconstruction with ectopic bone formation by GMP adipose stem cells.[J]. Int J Oral Maxillofac Surg.2009,38(3):201-209.
[63]De Ugarte D A, Alfonso Z, Zuk P A, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.[J]. Immunol Lett.2003,89(2-3):267-270.
[64]Oedayrajsingh-Varma M J, Van H S, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.[J]. Cytotherapy.2006,8(2): 166-177.
[65]Prunet-Marcassus B, Cousin B, Caton D, et al. From heterogeneity to plasticity in adipose tissues: site-specific differences.[J]. Exp Cell Res.2006,312(6): 727-736.
[66]Peptan I A, Hong L, Mao J J. Comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits.[J]. Plast Reconstr Surg. 2006,117(5):1462-1470.
[67]Mazo M, Planat-Benard V, Abizanda G, et al. Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction[J]. Eur J Heart Fail.2008,10(5):454-462.
[1]Migliaccio S, Brama M, Malavolta N. Management of glucocorticoids-induced osteoporosis:role of teriparatide[J]. Ther Clin Risk Manag.2009,5(2):305-310.
[2]Adler R A. Glucocorticoid-induced osteoporosis:management update[J]. Curr Osteoporos Rep.2010,8(1):10-14.
[3]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[4]Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J]. Tissue Eng.2001,7(2):211-228.
[5]邹丽宜,吴铁,崔燎,等.泼尼松致大鼠骨质疏松症的作用机制研究[J].中国药理学通报.2007,23(10)：1388-1392.
[6]史念珂,富东慧,蔡霞.大鼠股骨生物力学实验研究[J].天津大学学报.2006,39(4)：458-462.
[7]Parfitt A M, Drezner M K, Glorieux F H, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res.1987,2(6): 595-610.
[8]刘巍.TGF-p, IGF-1在骨质疏松骨折愈合过程中表达变化的实验研究[D].山西医科大学,2006.
[9]陈方经.鲑鱼降钙素对老年人股骨粗隆间骨折内固定术后的疗效评价[D].第二军医大学,2010.
[10]Dambacher M A, Olah A J, Maurer H, et al. [Pathogenesis, prevention and therapy of steroid osteoporosis][J]. Z Orthop Ihre Grenzgeb.1990,128(3): 234-239.
[11]Yao W, Cheng Z, Busse C, et al. Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis:a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice[J]. Arthritis Rheum.2008,58(6):1674-1686.
[12]De Nijs R N. Glucocorticoid-induced osteoporosis: a review on pathophysiology and treatment options[J]. Minerva Med.2008,99(1):23-43.
[13]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional Chinese medicine[J]. J Acupunct Meridian Stud.2010, 3(2):125-132.
[14]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional chinese medicine.[J]. J Acupunct Meridian Stud. 2010,3(2):125-132.
[15]白宇,原林,黄泳,等.经络的解剖学发现——筋膜学新理论[J].世界科学技术-中医药现代化.2010,12(1)：20-24.
[16]Lien C Y, Chih-Yuan H K, Lee O K, et al. Restoration of bone mass and strength in glucocorticoid-treated mice by systemic transplantation of CXCR4 and cbfa-1 co-expressing mesenchymal stem cells[J]. J Bone Miner Res.2009, 24(5):837-848.
[17]Mclaughlin F, Mackintosh J, Hayes B P, et al. Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers[J]. Bone.2002,30(6):924-930.
[18]Lukert B P, Raisz L G. Glucocorticoid-induced osteoporosis:pathogenesis and management[J]. Ann Intern Med.1990,112(5):352-364.
[19]Kozai Y, Kawamata R, Sakurai T, et al. Influence of prednisolone-induced osteoporosis on bone mass and bone quality of the mandible in rats[J]. Dentomaxillofac Radiol.2009,38(1):34-41.
[20]邹世恩,张绍芬,蒋莉,等.大豆苷原防治去卵巢大鼠骨质疏松症的量效作用[J].中国骨质疏松杂志.2008,14(3)：177-182.
[21]Griffith J F, Engelke K, Genant H K. Looking beyond bone mineral density: Imaging assessment of bone quality[J]. Ann N Y Acad Sci.2010,1192:45-56.
[22]孙平刘玲萍李捷.补肾壮骨中药对糖皮质激素诱发骨质疏松大鼠的干预作用[J].中药材.2010,33(04)：593-595.
[23]Kulak C A, Dempster D W. Bone histomorphometry: a concise review for endocrinologists and clinicians[J]. Arq Bras Endocrinol Metabol.2010,54(2): 87-98.
[24]Chavassieux P M, Arlot M E, Reda C, et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis[J]. J Clin Invest.1997,100(6):1475-1480.
[25]Thompson D D, Simmons H A, Pirie C M, et al. FDA Guidelines and animal models for osteoporosis [J]. Bone.1995,17(4 Suppl):125S-133S.
[26]李根林.骨松康胶囊对去卵巢骨质疏松大鼠骨生物力学性能的影响[D].河南中医学院,2007.
[27]Lee G H, Hwang J D, Choi J Y, et al. An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts:the involvement of BAX inhibitor-1[J]. Int J Biochem Cell Biol.2011,43(9):1305-1317.
[28]Parfitt A M. Renal bone disease:a new conceptual framework for the interpretation of bone histomorphometry[J]. Curr Opin Nephrol Hypertens. 2003,12(4):387-403.
[29]张仕兵.健骨片对去卵巢骨质疏松模型大鼠骨代谢影响的实验研究[D].南京中医药大学,2009.
[30]尹宏,钱卫庆.磷酸钙骨水泥-淫羊藿复合材料促进骨质疏松大鼠骨折的愈合[J].中国组织工程研究.2012,16(29)：5389-5395.
[31]贾经汉,邱新建,陈志坚.常用骨质疏松动物模型特点的综述[J].广西中医学院学报.2006,9(4)：76-79.
[2]贾经汉,邱新建,陈志坚.骨质疏松动物模型的研究进展[J].中国康复医学杂志.2007,22(8)：765-768.
[3]尚庆,刘宗超,鲁晓波.骨质疏松动物模型特点的综述[J].中国实用医药.2008,3(13)：193-195.
[4]何冬丽,米淑筠.骨质疏松症治疗及预防进展(综述)[J].天津护理.2001,9(5)：259-261.
[5]崔双,薛昊罡.绝经后女性骨质疏松患者骨密度与血脂和护骨素的相关性[J].中国老年学杂志.2012,32(10)：2051-2053.
[6]于文广,张鲁阳,张学顺.类风湿关节炎患者骨质疏松健康教育相关因素调查分析[J].中国骨质疏松杂志.2012,18(7)：623-627.
[7]邓娟,裴必伟,徐胜前,等.类风湿关节炎患者全身骨质疏松和局部骨侵蚀的关系研究[J].中华风湿病学杂志.2012,16(10)：674-678.
[8]唐鹏,孟国林,王军,等.长期应用双膦酸盐类药物对老年性骨质疏松骨折愈合过程的影响[J].中国骨质疏松杂志.2012,18(4)：317-322.
[9]刘刚,唐瑭,刘晶晶.阿仑膦酸钠联合钙尔奇D治疗老年骨质疏松62例[J].中国老年学杂志.2012,32(18)：4070-4071.
[10]高天君,杨达宇.比较切开复位内固定和石膏固定治疗老年骨质疏松性桡骨远端骨折对患肢继发性骨质疏松和关节功能的影响[J].中国骨质疏松杂志.2012,18(3)：245-247.
[11]郭庆华.老年性骨质疏松症药物治疗的新进展(综述)[J].中国城乡企业卫生.2004(4)：25-26.
[12]邓海鸥,李东风,张伟杰,等.静脉用唑来膦酸治疗老年骨质疏松患者时引起的急性期发热反应分析[J].南方医科大学学报.2011,31(12)：2076-2078.
[13]汪元浚,杨发满,李蓉,等.综合营养干预措施对老年原发性骨质疏松患者骨密度的影响[J].中国老年学杂志.2012,32(4)：727-728.
[14]高俊,张前德,张曦,等.废用性骨质疏松动物模型的建立与评价[J].广东医学.2012,33(18)：2728-2731.
[15]廖立,杨小红,金岩.骨质疏松发病过程中骨髓间充质干细胞差异性表达microRNA的筛选及其在干细胞多向分化中的功能[J].浙江大学学报(医学版).2012,41(1)：75-80.
[16]袁绍辉,刘伟,吴滨奇,等.骨质疏松性骨折愈合中骨改建的动态参数及骨密度变化[J].中国组织工程研究与临床康复.2011,15(41)：7627-7630.
[17]孙允高,罗先正,阮祥燕,等.骨质疏松诊断技术的新进展[J].仪器仪表学报.2000,21(Z1)：25-27,40.
[18]钱慕周,尹友生,何勇,等.雷洛昔芬对血液透析妇女骨质疏松的干预作用[J].山东医药.2012,52(12)：83-85.
[19]项丽娟,张艳,庞炜.运动干预骨质疏松大鼠对二甲基精氨酸二甲胺水解酶表达的影响[J].山东体育学院学报.2011,27(8)：62-65.
[20]成强.优选组合用药治疗老年骨质疏松的疗效[J].中国老年学杂志.2012,32(15)：3318-3320.
[21]单光宇,薛昊罡.糖尿病骨质疏松大鼠AGEs的表达及其与TGF-p1的相关性[J].中国老年学杂志.2012,32(11)：2344-2345.
[22]任炼,李志忠,隋杰,等.骨水泥充填椎体成形治疗骨质疏松性椎体压缩骨折：6年随访[J].中国组织工程研究.2012,16(12)：2110-2114.
[23]杨宁,崔岳毅,韩晓光,等.骨质疏松大鼠股骨内单次注射辛伐他汀强化骨骼的实验研究[J].中华骨科杂志.2012,32(5)：500-506.
[24]范光磊,薄静莉,陈杰敏,等.骨转换生化标志物与老年2型糖尿病患者并发骨质疏松的相关性研究[J].中华老年医学杂志.2012,31(8)：657-661.
[25]杨希重,季翠杰,王德春,等.去卵巢骨质疏松模型大鼠的番茄红素干预[J].中国组织工程研究.2012,16(15)：2750-2756.
[26]刘少军,张娴,冯丽敏,等.应力缺失致骨质疏松大鼠成骨细胞细胞信号外调节激酶表达及通络生骨胶囊的影响[J].中国组织工程研究与临床康复.2011,15(20)：3625-3628.
[1]Migliaccio S, Brama M, Malavolta N. Management of glucocorticoids-induced osteoporosis: role of teriparatide[J]. Ther Clin Risk Manag.2009,5(2):305-310.
[2]Adler R A. Glucocorticoid-induced osteoporosis:management update[J]. Curr Osteoporos Rep.2010,8(1):10-14.
[3]王迎军,杜昶,赵娜如,等.仿生人工骨修复材料研究[J].华南理工大学学报(自然科学版).2012,40(10)：51-58.
[4]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[5]许宋锋,胡蕴玉,林开利,等.多孔硅酸钙生物陶瓷体内非骨性环境的植入研究[J].无机材料学报.2008,23(3)：611-616.
[6]刘冰,李淑娟,杨冬茹,等.大黄素治疗实验性牙周炎的骨计量学研究[J].实用口腔医学杂志.2010,26(5)：593-596.
[7]Parfitt A M, Drezner M K, Glorieux F H, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res.1987,2(6): 595-610.
[8]王学英.骨质疏松症防治药物发展现状[J].青海医药杂志.2008,38(11)：75-77.
[9]尚庆,刘宗超,鲁晓波.骨质疏松动物模型特点的综述[J].中国实用医药.2008,3(13)：193-195.
[10]陈璐,周文清,吴燕平,等.p-磷酸三钙复合自体骨移植在上颌窦闭合式提 升中的应用[J].上海口腔医学.2011,20(3)：282-285.
[11]姚志鹏,刘文革,倪国新.掺锶羟基磷灰石骨水泥的生物学特点及在骨科的应用[J].中国组织工程研究与临床康复.2008,12(36)：7151-7154.
[12]Kulak C A, Dempster D W. Bone histomorphometry:a concise review for endocrinologists and clinicians[J]. Arq Bras Endocrinol Metabol.2010,54(2): 87-98.
[13]Chavassieux P M, Arlot M E, Reda C, et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis[J]. J Clin Invest.1997,100(6):1475-1480.
[14]Thompson D D, Simmons H A, Pirie C M, et al. FDA Guidelines and animal models for osteoporosis[J]. Bone.1995,17(4 Suppl):125S-133S.
[15]Parfitt A M. Renal bone disease: a new conceptual framework for the interpretation of bone histomorphometry[J]. Curr Opin Nephrol Hypertens. 2003,12(4):387-403.
[16]裴玉岩,刘继光,李凌志,等.淫羊藿对骨质疏松家兔颌骨骨缺损愈合的影响[J].黑龙江医药科学.2011,34(1)：93-94.
[17]王世惟,裴庆国,宋萌.骨髓基质干细胞与β-TCP生物相容性的实验研究[J].上海口腔医学.2010,19(3)：319-322.
[18]沈宇辉,邓廉夫,潘浩波,等.含锶可降解生物陶瓷及生物玻璃骨植入材料研究进展[J].国际骨科学杂志.2011,32(2)：75-77.
[19]李峰,赵信义,吴军正,等.含锶磷酸钙骨水泥生物相容性评价[J].牙体牙髓牙周病学杂志.2006,16(5)：264-268.
[20]朱磊.微量元素在骨质疏松防治中的作用[J].上海医药.2012,33(8)：43-46.
[21]Lee G H, Hwang J D, Choi J Y, et al. An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts:the involvement of BAX inhibitor-1[J]. Int J Biochem Cell Biol.2011,43(9):1305-1317.
[22]袁月,哈斯达来,张劲松,等.碳化硅对成骨细胞增殖和分化的影响[J].生物医学工程与临床.2012,16(2)：109-113.
[1]Trzaska K A, Castillo M D, Rameshwar P. Adult mesenchymal stem cells in neural regeneration and repair: Current advances and future prospects (Review)[J]. Mol Med Report.2008,1(3):307-316.
[2]Augello A, Kurth T B, De Bari C. Mesenchymal stem cells:a perspective from in vitro cultures to in vivo migration and niches[J]. Eur Cell Mater.2010,20: 121-133.
[3]Kogler G, Sensken S, Wernet P. Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood[J]. Exp Hematol.2006,34(11):1589-1595.
[4]Fumimoto Y, Matsuyama A, Komoda H, et al. Creation of a rich subcutaneous vascular network with implanted adipose tissue-derived stromal cells and adipose tissue enhances subcutaneous grafting of islets in diabetic mice[J]. Tissue Eng Part C Methods.2009,15(3):437-444.
[5]Xiao Y, Mareddy S, Crawford R. Clonal characterization of bone marrow derived stem cells and their application for bone regeneration[J]. Int J Oral Sci. 2010,2(3):127-135.
[6]孙慧如,王英,董明敏.豚鼠脂肪干细胞的分离培养与鉴定[J].中国组织工程研究与临床康复.2007,11(7)：1213-1215.
[7]Han Z B, Chen H X, Deng J X. [Multipotential differentiation and potential applications of adipose-derived stem cells][J]. Sheng Wu Gong Cheng Xue Bao.2007,23(2):195-200.
[8]韩长杰,杨向群,张传森.脂肪干细胞的免疫学性质[J].中国组织工程研究与临床康复.2010,14(27)：5095-5098.
[9]Nakao N, Nakayama T, Yahata T, et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal stem cells[J]. Am J Pathol.2010,177(2): 547-554.
[10]Mosna F, Sensebe L, Krampera M. Human bone marrow and adipose tissue mesenchymal stem cells:a user's guide[J]. Stem Cells Dev.2010,19(10): 1449-1470.
[11]Casteilla L, Planat-Benard V, Bourin P, et al. [Use of adipose tissue in regenerative medicine.][J]. Transfus Clin Biol.2011.
[12]de Mattos C A, Alves A L, Golim M A, et al. Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue.[J]. Vet Immunol Immunopathol.2009,132(2-4):303-306.
[13]Sung J H, Yang H M, Park J B, et al. Isolation and characterization of mouse mesenchymal stem cells.[J]. Transplant Proc.2008,40(8):2649-2654.
[14]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional Chinese medicine[J]. J Acupunct Meridian Stud. 2010,3(2):125-132.
[15]原林,钟世镇.人体自体检测与调控系统(筋膜学)--经络有关的解剖学基础[J].天津中医药.2004,21(5)：356-359.
[16]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[17]Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies[J]. Tissue Eng.2001,7(2): 211-228.
[18]Choi Y S, Cha S M, Lee Y Y, et al. Adipogenic differentiation of adipose tissue derived adult stem cells in nude mouse[J]. Biochem Biophys Res Commun.2006,345(2):631-637.
[19]Luzi E, Marini F, Sala S C, et al. Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor[J]. J Bone Miner Res.2008,23(2):287-295.
[20]Gregory C A, Gunn W G, Peister A, et al. An Alizarin red-based assay of mineralization by adherent cells in culture:comparison with cetylpyridinium chloride extraction[J]. Anal Biochem.2004,329(1):77-84.
[21]Katz A J, Tholpady A, Tholpady S S, et al. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells[J]. Stem Cells.2005,23(3):412-423.
[22]Wan D C, Shi Y Y, Nacamuli R P, et al. Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling[J]. Proc Natl Acad Sci U S A. 2006,103(33):12335-12340.
[23]Madonna R, De Caterina R. Adipose tissue: a new source for cardiovascular repair[J]. J Cardiovasc Med (Hagerstown).2010,11(2):71-80.
[24]Yanez R, Lamana M L, Garcia-Castro J, et al. Adipose tissue-derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft-versus-host disease[J]. Stem Cells.2006,24(11): 2582-2591.
[25]Vieira N M, Brandalise V, Zucconi E, et al. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells[J]. Cell Transplant.2010,19(3):279-289.
[26]Mcintosh K, Zvonic S, Garrett S, et al. The immunogenicity of human adipose-derived cells:temporal changes in vitro[J]. Stem Cells.2006,24(5): 1246-1253.
[27]Zhu Y, Liu T, Song K, et al. Adipose-derived stem cell:a better stem cell than BMSC[J]. Cell Biochem Funct.2008,26(6):664-675.
[28]Rigotti G, Marchi A, Sbarbati A. Adipose-derived mesenchymal stem cells: past, present, and future[J]. Aesthetic Plast Surg.2009,33(3):271-273.
[29]Ren Y, Wu H, Ma Y, et al. [Isolation, cultivation and identification of adipose-derived stem cell in bovines][J]. Sheng Wu Gong Cheng Xue Bao. 2010,26(12):1645-1651.
[30]Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential[J]. Cytotherapy.2003,5(5): 362-369.
[31]Schaffler A, Buchler C. Concise review:adipose tissue-derived stromal cells--basic and clinical implications for novel cell-based therapies.[J]. Stem Cells.2007,25(4):818-827.
[32]王栋,鹿均先,熊传芝.人脂肪干细胞分离培养鉴定及向成骨细胞诱导分化的实验研究[J].现代医学.2008,36(3)：190-193.
[33]Korn B S, Kikkawa D O, Hicok K C. Identification and characterization of adult stem cells from human orbital adipose tissue[J]. Ophthal Plast Reconstr Surg.2009,25(1):27-32.
[34]Gu F, Gao J H, Lu F. [Culture of the single cell clones of human adipose-derived stem cells and identification of their surface antigen expressions][J]. Nan Fang Yi Ke Da Xue Xue Bao.2008,28(6):1067-1069.
[35]Zola H, Swart B, Boumsell L, et al. Human Leucocyte Differentiation Antigen nomenclature:update on CD nomenclature. Report of IUIS/WHO Subcommittee.[J]. J Immunol Methods.2003,275(1-2):1-8.
[36]CD antigens 1996:updated nomenclature for clusters of differentiation on human cells. IUIS/WHO Subcommittee on CD Nomenclature.[J]. Bull World Health Organ.1997,75(4):385-387.
[37]Fukumori T, Takenaka Y, Yoshii T, et al. CD29 and CD7 mediate galectin-3-induced type II T-cell apoptosis.[J]. Cancer Res.2003,63(23): 8302-8311.
[38]Munakata Y, Saito T, Watanabe T, et al. Rapid inhibitory effect of tacrolimus on T cell migration by suppressing CD29-related functions.[J]. Clin Exp Rheumatol.2004,22(2):197-204.
[39]Rizzatti E G, Garcia A B, Portieres F L, et al. Expression of CD117 and CDllb in bone marrow can differentiate acute promyelocytic leukemia from recovering benign myeloid proliferation.[J]. Am J Clin Pathol.2002,118(1): 31-37.
[40]Paulli M, Rosso R, Kindl S, et al. Immunophenotypic characterization of the cell infiltrate in five cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease).[J]. Hum Pathol.1992,23(6): 647-654.
[41]Shaw E B. Visibility amplification of intestinal intraepithelial lymphocytes.[J]. Arch Pathol Lab Med.2002,126(8):897.
[42]Krasinskas A M, Wasik M A, Kamoun M, et al. The usefulness of CD64, other monocyte-associated antigens, and CD45 gating in the subclassification of acute myeloid leukemias with monocytic differentiation.[J]. Am J Clin Pathol. 1998,110(6):797-805.
[43]Chesney J, Metz C, Stavitsky A B, et al. Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes.[J]. J Immunol.1998,160(1):419-425.
[44]Alon R, Feigelson S W, Manevich E, et al. Alpha4betal-dependent adhesion strengthening under mechanical strain is regulated by paxillin association with the alpha4-cytoplasmic domain.[J]. J Cell Biol.2005,171(6):1073-1084.
[45]Soilu-Hanninen M, Laaksonen M, Hanninen A. Hyaluronate receptor (CD44) and integrin alpha4 (CD49d) are up-regulated on T cells during MS relapses.[J]. J Neuroimmunol.2005,166(1-2):189-192.
[46]Miller D H, Khan O A, Sheremata W A, et al. A controlled trial of natalizumab for relapsing multiple sclerosis.[J]. N Engl J Med.2003,348(1):15-23.
[47]Berger J R, Houff S. Progressive multifocal leukoencephalopathy:lessons from AIDS and natalizumab.[J]. Neurol Res.2006,28(3):299-305.
[48]Negrin R S, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34+Thy-1+hematopoietic stem cells in patients with metastatic breast cancer.[J]. Biol Blood Marrow Transplant.2000,6(3):262-271.
[49]Wetzel A, Chavakis T, Preissner K T, et al. Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD1 lb/CD18).[J]. J Immunol.2004,172(6):3850-3859.
[50]Nakamura Y, Muguruma Y, Yahata T, et al. Expression of CD90 on keratinocyte stem/progenitor cells.[J]. Br J Dermatol.2006,154(6):1062-1070.
[51]Chang A, Benda P M, Wood B L, et al. Lineage-specific identification of nonhematopoietic neoplasms by flow cytometry.[J]. Am J Clin Pathol.2003, 119(5):643-655.
[52]Johnson L A, Clasper S, Holt A P, et al. An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium.[J]. J Exp Med.2006,203(12):2763-2777.
[53]Cybulsky M I, Iiyama K, Li H, et al. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis.[J]. J Clin Invest.2001,107(10):1255-1262.
[54]Barrier B F, Sharpe-Timms K L. Expression of soluble adhesion molecules in sera of women with stage III and IV endometriosis.[J]. J Soc Gynecol Investig. 2002,9(2):98-101.
[55]Cardier J E, Rivas B, Romano E, et al. Evidence of vascular damage in dengue disease:demonstration of high levels of soluble cell adhesion molecules and circulating endothelial cells.[J]. Endothelium.2006,13(5):335-340.
[56]Silva H C, Garcao F, Coutinho E C, et al. Soluble VCAM-1 and E-selectin in breast cancer: relationship with staging and with the detection of circulating cancer cells.[J]. Neoplasma.2006,53(6):538-543.
[57]Kim U, Shin D G, Park J S, et al. Homing of adipose-derived stem cells to radiofrequency catheter ablated canine atrium and differentiation into cardiomyocyte-like cells.[J]. Int J Cardiol.2009.
[58]Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells.[J]. Biochem Biophys Res Commun.2002,294(2):371-379.
[59]雷磊,廖威明,盛璞义,等.人类脂肪来源成体干细胞体外培养的生物学特征研究及供体年龄对其增殖的影响[J].中国科学(C辑：生命科学).2007(02).
[60]Wagner W, Wein F, Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood.[J]. Exp Hematol.2005,33(11):1402-1416.
[61]De U D, Alfonso Z, Zuk P A, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.[J]. Immunol Lett.2003,89(2-3):267-270.
[62]Mesimaki K, Lindroos B, Tornwall J, et al. Novel maxillary reconstruction with ectopic bone formation by GMP adipose stem cells.[J]. Int J Oral Maxillofac Surg.2009,38(3):201-209.
[63]De Ugarte D A, Alfonso Z, Zuk P A, et al. Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.[J]. Immunol Lett.2003,89(2-3):267-270.
[64]Oedayrajsingh-Varma M J, Van H S, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.[J]. Cytotherapy.2006,8(2): 166-177.
[65]Prunet-Marcassus B, Cousin B, Caton D, et al. From heterogeneity to plasticity in adipose tissues: site-specific differences.[J]. Exp Cell Res.2006,312(6): 727-736.
[66]Peptan I A, Hong L, Mao J J. Comparison of osteogenic potentials of visceral and subcutaneous adipose-derived cells of rabbits.[J]. Plast Reconstr Surg. 2006,117(5):1462-1470.
[67]Mazo M, Planat-Benard V, Abizanda G, et al. Transplantation of adipose derived stromal cells is associated with functional improvement in a rat model of chronic myocardial infarction[J]. Eur J Heart Fail.2008,10(5):454-462.
[1]Migliaccio S, Brama M, Malavolta N. Management of glucocorticoids-induced osteoporosis:role of teriparatide[J]. Ther Clin Risk Manag.2009,5(2):305-310.
[2]Adler R A. Glucocorticoid-induced osteoporosis:management update[J]. Curr Osteoporos Rep.2010,8(1):10-14.
[3]杨春,李东飞,戴景兴,等.异体脂肪源干细胞移植对大鼠的抗衰老作用[J].解剖学报.2010,41(1)：87-92.
[4]Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J]. Tissue Eng.2001,7(2):211-228.
[5]邹丽宜,吴铁,崔燎,等.泼尼松致大鼠骨质疏松症的作用机制研究[J].中国药理学通报.2007,23(10)：1388-1392.
[6]史念珂,富东慧,蔡霞.大鼠股骨生物力学实验研究[J].天津大学学报.2006,39(4)：458-462.
[7]Parfitt A M, Drezner M K, Glorieux F H, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee[J]. J Bone Miner Res.1987,2(6): 595-610.
[8]刘巍.TGF-p, IGF-1在骨质疏松骨折愈合过程中表达变化的实验研究[D].山西医科大学,2006.
[9]陈方经.鲑鱼降钙素对老年人股骨粗隆间骨折内固定术后的疗效评价[D].第二军医大学,2010.
[10]Dambacher M A, Olah A J, Maurer H, et al. [Pathogenesis, prevention and therapy of steroid osteoporosis][J]. Z Orthop Ihre Grenzgeb.1990,128(3): 234-239.
[11]Yao W, Cheng Z, Busse C, et al. Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis:a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice[J]. Arthritis Rheum.2008,58(6):1674-1686.
[12]De Nijs R N. Glucocorticoid-induced osteoporosis: a review on pathophysiology and treatment options[J]. Minerva Med.2008,99(1):23-43.
[13]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional Chinese medicine[J]. J Acupunct Meridian Stud.2010, 3(2):125-132.
[14]Bai Y, Yuan L, Soh K S, et al. Possible applications for fascial anatomy and fasciaology in traditional chinese medicine.[J]. J Acupunct Meridian Stud. 2010,3(2):125-132.
[15]白宇,原林,黄泳,等.经络的解剖学发现——筋膜学新理论[J].世界科学技术-中医药现代化.2010,12(1)：20-24.
[16]Lien C Y, Chih-Yuan H K, Lee O K, et al. Restoration of bone mass and strength in glucocorticoid-treated mice by systemic transplantation of CXCR4 and cbfa-1 co-expressing mesenchymal stem cells[J]. J Bone Miner Res.2009, 24(5):837-848.
[17]Mclaughlin F, Mackintosh J, Hayes B P, et al. Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers[J]. Bone.2002,30(6):924-930.
[18]Lukert B P, Raisz L G. Glucocorticoid-induced osteoporosis:pathogenesis and management[J]. Ann Intern Med.1990,112(5):352-364.
[19]Kozai Y, Kawamata R, Sakurai T, et al. Influence of prednisolone-induced osteoporosis on bone mass and bone quality of the mandible in rats[J]. Dentomaxillofac Radiol.2009,38(1):34-41.
[20]邹世恩,张绍芬,蒋莉,等.大豆苷原防治去卵巢大鼠骨质疏松症的量效作用[J].中国骨质疏松杂志.2008,14(3)：177-182.
[21]Griffith J F, Engelke K, Genant H K. Looking beyond bone mineral density: Imaging assessment of bone quality[J]. Ann N Y Acad Sci.2010,1192:45-56.
[22]孙平刘玲萍李捷.补肾壮骨中药对糖皮质激素诱发骨质疏松大鼠的干预作用[J].中药材.2010,33(04)：593-595.
[23]Kulak C A, Dempster D W. Bone histomorphometry: a concise review for endocrinologists and clinicians[J]. Arq Bras Endocrinol Metabol.2010,54(2): 87-98.
[24]Chavassieux P M, Arlot M E, Reda C, et al. Histomorphometric assessment of the long-term effects of alendronate on bone quality and remodeling in patients with osteoporosis[J]. J Clin Invest.1997,100(6):1475-1480.
[25]Thompson D D, Simmons H A, Pirie C M, et al. FDA Guidelines and animal models for osteoporosis [J]. Bone.1995,17(4 Suppl):125S-133S.
[26]李根林.骨松康胶囊对去卵巢骨质疏松大鼠骨生物力学性能的影响[D].河南中医学院,2007.
[27]Lee G H, Hwang J D, Choi J Y, et al. An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts:the involvement of BAX inhibitor-1[J]. Int J Biochem Cell Biol.2011,43(9):1305-1317.
[28]Parfitt A M. Renal bone disease:a new conceptual framework for the interpretation of bone histomorphometry[J]. Curr Opin Nephrol Hypertens. 2003,12(4):387-403.
[29]张仕兵.健骨片对去卵巢骨质疏松模型大鼠骨代谢影响的实验研究[D].南京中医药大学,2009.
[30]尹宏,钱卫庆.磷酸钙骨水泥-淫羊藿复合材料促进骨质疏松大鼠骨折的愈合[J].中国组织工程研究.2012,16(29)：5389-5395.
[31]贾经汉,邱新建,陈志坚.常用骨质疏松动物模型特点的综述[J].广西中医学院学报.2006,9(4)：76-79.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |