外源性脂肪源干细胞移植改善D-半乳糖致衰老大鼠性能力的研究
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摘要
背景：
衰老(senescence)又称老化,是整个机体形态结构和生理机能的全面衰退,是一个动态的、复杂的过程。当前预防和延缓衰老已成为生命科学研究的热点。有关衰老机制的研究一直是生物学、老年医学研究的前沿课题。对其机制的研究中有自由基学说、代谢学说、免疫系统退化学说、端粒酶变化等多种学说,但没有一种学说能完整解释衰老的机制。
原林教授提出的“筋膜学说”认为：人体由两部分组成,一个是由尚未分化的非特异性结缔组织(筋膜)支架所构成的支持与储备系统,一个是以已分化的功能细胞为基础的功能系统。功能系统在支持与储备系统支持下维持结构与功能的稳定,而支持与储备系统源源不断的为功能系统的细胞更新和功能活动提供细胞供应和营养物质。功能系统是以功能细胞的专能特化为基础的,而支持与储备系统的筋膜支架以干细胞为核心,通过分化出各种定向干细胞,为功能系统的更新提供源源不断的细胞补充,并为功能系统的各种细胞的更新、代谢提供一个稳定的内部环境。即衰老是筋膜中干细胞储备逐渐耗竭的过程,而如何保持筋膜的正常状态,从而为功能系统提供稳定的细胞来源并维持其向功能细胞的正常分化,是延长生命周期的关键。如果衰老时减少的干细胞能得到及时的补充,为机体提供新的细胞储备,可修复机体损伤,延缓衰老、延长生命周期。不同于其他的衰老学说,筋膜学说从另一个不同的角度——发育生物学——对衰老提出了新的认识,同时与从微观角度提出的各种衰老学说并无矛盾,认为各种微观角度的改变最终导致的结果都是筋膜组织—支持与储备系统功能的减退。
间充质干细胞(menchymal stem cells)具有自我更新、多向分化的能力。最初是由Friendenstein等发现,而且证明其在体外可以分化成为成骨细胞及脂肪细胞,而后其它的研究小组发现骨髓来源的MSCs不仅可以分化为中胚层来源的组织,而且可以分化为内胚层和外胚层来源的组织。而且间充质干细胞的多组织来源与筋膜结缔组织支架的全身分布是一致的,其纵向、横向分化的生物学特性与筋膜学提出的筋膜结缔组织对机体的支持储备作用也是一致的。脂肪源干细胞(ADSCs)是筋膜中未分化干细胞的一种储备形式。ADSCs具有多向分化潜能,能够跨胚层分化。它可以分化成中胚层来源的脂肪细胞、成骨细胞、软骨细胞、心肌细胞；外胚层来源的神经细胞；也可以分化成内胚层来源的内皮细胞及肝细胞等。ADSCs具有低免疫原性,免疫调节功能和分泌细胞因子等能力,为异体移植提供了有利条件,使ADSCs成为生物治疗的亮点之一；ADSCs分泌细胞因子抗老化的作用,已经被临床广泛应用。ADSCs不仅可以用来进行各种退行性和衰竭疑难病的替代治疗,而且可用来进行辅助的免疫治疗。脂肪源干细胞可以调节受损组织局部微环境,可以分泌一系列细胞因子和生长因子到周围组织液中,调节组织微环境,形成有利于干细胞生长的微环境--“干细胞巢”--招募来自于受体的内源性干细胞到达靶点,并使其向所需要的特定组织细胞定向分化。脂肪干细胞本身能够耐受缺氧,同时在缺氧的环境中还能释放抗氧化物质、自由基清除剂、分子伴侣、热休克蛋白等,清除受损局部的细胞毒性物质,促进尚存活的细胞复苏,在脂肪移植后形成有利于组织细胞存活和功能维持的微环境。ADSC还有可能通过旁分泌细胞因子促进组织修复。经静脉注射的ADSC可自主聚集于病灶部位参与修复损伤。ADSC的这种“归巢”作用,其具体机制不明。有报道称通过增加膜型基质金属蛋白酶1(membranetype1matrix metallop roteinase, MT1-MMP)表达和促进MMP-2酶原的激活可增强ADSCs的迁移。近期研究发现,ADSC能释放新的线粒体至损害的细胞,从而挽救有氧代谢的过程。这说明ADSC可通过多种机制参与组织的修复。
简言之,ADSCs可能通过以下机制修复或者重建组织：首先,ADSCs被植入一个受损的或是坏死的组织,它们能以副分泌的方式分泌出细胞活素和生长因子以刺激修复。ADSCs可以提供抗氧化剂、自由基清除剂、热休克蛋白等给待修复组织,从而清除局部环境中所释放的有毒物质,促进仍存活的细胞的修复。另外,ADSCs可以通过刺激内生性干细胞补充到待修复的脏器或自身直接迁移补充到待修复区域,以调节干细胞的微环境,并且促进它们沿着需要的方式分化为功能细胞,从而更新机体衰老的功能细胞,维持机体平衡。
D-gal所致亚急性衰老模型是国内外比较公认的衰老模型。在一定时间内连续给动物注射D-半乳糖使机体细胞糖代谢及脂代谢紊乱,体内糖浓度增高后被醛糖还原酶催化还原成半乳糖醇,其不能被细胞进一步代谢而堆积体内、影响正常渗透压,导致细胞肿胀、功能障碍、代谢紊乱,不仅破坏并消耗机体抗氧化防御系统,使自由基积聚；同时在半乳糖还原成半乳糖醇的过程中又使细胞膜脂质受损,过氧化脂质、脂褐素增高,出现衰老。糖代谢紊乱必然会引起心、肝、肾、脑等重要器官代谢异常,最后出现衰老。D-半乳糖模型因全面影响细胞的代谢功能和一些重要的酶的功能,所导致的老化较全面。其老化与机能减退的具体机制可能与代谢障碍、免疫损伤、自由基损伤及线粒体损伤等相关。目前,该模型已广泛应用于研究衰老机制以及延缓衰老药物的筛选。D-gal致亚急性衰老模型可以模拟脑、肝脏、肾脏、皮肤、性腺轴、骨质疏松、免疫系统等多脏器的损伤。文献报道可应用干细胞移植治疗,亦有文献报道应用中药提取物、中药汤方、激素及针灸等多种方法治疗此模型造成的衰老。
雄激素可提高性中枢神经系统的兴奋性,诱发性欲冲动,从而维持正常的性机能。睾丸间质细胞(Leydig cells)是机体分泌睾酮的主要细胞,分泌量约占身体总睾酮的95%；国际上对男性生殖疾病治疗的研究也多集中在Leydig细胞的培养和移植上。睾丸间质细胞具有合成、分泌睾酮的特点。睾酮合成过程中,17β-HSD3、3p-HSD1催化脱氢异雄酮形成睾酮。而3β-HSD是合成睾酮的关键酶,成年大鼠睾丸中只有间质细胞表达3β-HSD,所以目前对间质细胞的鉴定一般都采用3β-HSD特异性染色方法。Percoll梯度浓度分离的Leydig细胞量大、纯度较高、细胞活力良好,初步解决了组织工程技术构建雄激素分泌组织的种子细胞来源的问题。用该方法获得的Leydig细胞所构建的组织工程化雄激素分泌组织能在较长时期维持睾酮分泌功能。
为探讨外源性脂肪源干细胞移植是否具有延缓衰老的作用及其作用机制,本实验体内观察移植外源性ADSCs对D-半乳糖(D-gal)衰老大鼠自由基及免疫等衰老指标,以及对衰老大鼠性能力的改善等方面的影响；体外观察脂肪源干细胞基质与D-gal处理的睾丸间质细胞(LCs)共培养,探讨ADSCs对Leydig cell的保护性作用,探索一种新的抗衰老机理,为临床延缓衰老的治疗提供新的思路和方法。
目的
研究移植脂肪源干细胞对D-半乳糖致衰老大鼠相关衰老指标以及交配能力的影响,探讨临床替代疗法的治疗机理,进一步为筋膜结缔组织是干细胞的储备源泉提供证据支持,为筋膜学提供理论支撑。
1、检验非特异性筋膜结缔组织中是否存在脂肪源干细胞。
2、观察移植脂肪源干细胞能否对D-半乳糖衰老大鼠的血清、自由基及免疫功能产生影响,阐述移植脂肪源干细胞对抗D-半乳糖衰老的血清学意义。
3、通过观察交配实验,研究移植筋膜结缔组织中的脂肪源干细胞能否提高D-半乳糖致衰老大鼠的性能力；通过免疫组织化学染色,观察移植标记的脂肪源干细胞在睾丸组织中的定位和移位情况,及睾丸间质细胞分泌3p-HSD、17β-HSD的情况；通过免疫组织化学染色,观察睾丸间质细胞细胞形态学变化及其凋亡状况；通过血清学检测,观察睾酮分泌状况。
4、体外分离培养睾丸间质细胞,观察脂肪干细胞对D-gal处理的睾丸间质细胞的保护性作用,为“筋膜学”抗衰老相关理论提供进一步佐证。
方法
1、取成年SD大鼠腹股沟脂肪垫,用酶消化法分离、培养筋膜结缔组织的脂肪源干细胞。通过用形态学、功能学、流式细胞术鉴定非特异性筋膜结缔组织源细胞是否符合间充质干细胞的特性,进而判断非特异性筋膜结缔组织中是否含有间充质干细胞。
2、体外Percoll分离液梯度分离培养睾丸间质细胞,并通过形态学及3β-HSD特异性染色鉴定；以0、4、8、12、16g/L不同浓度的D-gal处理睾丸间质细胞,每个浓度做三个复孔,每个孔重复三次。24h后,计算细胞存活率,筛选最适浓度模拟细胞衰老模型；另流式细胞仪检测8g/LD-半乳糖处理Leydig cell在0、24、48、72h不同时间点的细胞周期变化。
3、体外分离培养4代ADSCs,取上清制备脂肪干细胞基质(ADSC-CM);8g/LD-半乳糖处理Leydig cell,同时与ADSC-CM共培养24h后,MTT检测其细胞活性；流式细胞仪检测各组细胞共培养24h的周期变化；共培养48h,SA-p-半乳糖酐酶染色,每组取4个视野,每个视野至少100个细胞。检测细胞衰老情况；免疫组化观察3p-HSD分泌情况。
4、30只SD大鼠随机分成空白对照组(A组)、模型组(B组)和治疗组(C组),每组10只。B、C组颈背部皮下注射15%D-半乳糖1000mg/(kg-d),连续8周复制亚急性衰老模型,A组的大鼠每天给予生理盐水1.6ml颈背部皮下注射。
5、模拟衰老模型成功后,治疗组内的大鼠进行脂肪源干细胞静脉移植：移植的第3代ADSCs在进行传代后,在培养基中掺入10μmol/L的Brdu,孵育3天后消化收集,无菌生理盐水重悬,制成300万/ml；同样每只大鼠经尾静脉注射ADSCs3×106个,模型组输入生理盐水1ml。
6、交配实验的雌鼠分别在实验前48h和4h采用皮下注射苯甲酸雌二醇200μg/kg和黄体酮2mg/kg用来诱导雌鼠发情,用发情的雌鼠和雄鼠进行交配,从而对雄性进行相关性能力研究。HE染色显示大鼠睾丸组织精曲小管结构变化,采用免疫组化及免疫荧光追踪移植细胞；免疫组化观察3β-HSD1、17β-HSD3分泌情况,TUNEL免疫组化染色观察大鼠间质细胞凋亡指数。放射免疫法观察大鼠血清睾酮含量。
7、采用SPSS13.0统计软件处理,以均数±标准差(x±s)表示,方差齐时,多样本均数比较均采用单因素方差分析,两两比较采用LSD法；方差不齐时,多样本均数比较均采用Welch法,两两比较采用Dunnett's T3方法,P<0.05表示有统计学差异。
结果
1、腹股沟皮下筋膜结缔组织内的脂肪源干细胞具有长梭形、多角形的形态学特征,可形成细胞集落,体外可以诱导分化为成骨细胞、脂肪细胞。
2、体外Percoll分离法可成功分离培养睾丸间质细胞。间质细胞呈多边形、三角形。单层贴壁生长,伸展后呈漩涡状排列,细胞以集落形式增殖；经3p-HSD特异性染色鉴定,镜下可见睾丸间质细胞被染成蓝黑色而其他睾丸细胞不着色,睾丸间质细胞纯度可达到95%以上；MTT法显示8g/L的D-gal模拟衰老模型细胞存活率为(81.96±0.96),为最适浓度,可成功诱导leydig cell老化(F=8960.772,P=0.000)；且随时间延长,8g/L的D-gal处理的LCs呈现G0/G1期细胞增多,S期细胞减少。
3、ADSCs上清中可以提取出ADSCs-CM。空白对照组、8g/LD-gal处理的Leydig cells组、8g/LD-gal处理的Leydig cells与ADSCs-CM共培养组做比较,三组之间差异有统计学意义(F=1101.980,P<0.001)。MTT检测显示其细胞活性有所提高(P<0.001)；流式细胞仪共培养24h后,细胞周期中G0/G1期细胞比例下降；
空白对照组、8g/LD-gal处理的Leydig cells组、共培养组之间的SA-β-半乳糖酐酶阳性细胞数差异有统计学意义(F=1105.307,P<0.001)；共培养48h, SA-β-半乳糖酐酶染色,共培养组SA-β-半乳糖酐酶阳性细胞减少(P<0.001),细胞衰老减轻；免疫组化观察共培养后细胞分泌的3β-HSD分泌情况增加。
4、连续皮下注射D-gal可以模拟大鼠衰老。空白对照组、D-gal处理组、ADSCs-移植组之间的CuZn-SOD (F=28.587, P<0.001)、NO水平(F=56.571,P<0.001)、血清IL-2水平(F=23.667,P<0.001)、脾脏指数(F=33.830,P<0.001)和胸腺指数(F=15.847, P<0.001)、MDA水平(F=45.468,P<0.001)差异有统计学意义。衰老模型组与空白对照组比较,血清MDA水平有所升高(P<0.001),血清CuZn-SOD(P<0.001)、血清NO(P<0.001、IL-2水平(P<0.001)以及脾脏指数(P<0.001)、胸腺指数(P<0.001)均有所降低,提示D-半乳糖模拟亚急性大鼠衰老模型复制成功。与模型组比较,细胞治疗后衰老大鼠SOD活性(P=0.04)、血清IL-2水平(p=0.038)、血清NO水平(P<0.001)和胸腺指数(P=0.002)、脾脏指数(P=0.001)均有所升高,MDA水平则降低(P<0.001)。
5、空白对照组、D-gal模型处理组、ADSCs-移植组之间的血清睾酮含量比较差异有统计学意义(F=25.370,P<0.001),与空白对照组比较,模型组血清睾酮含量有所降低(P<0.001),提示D-半乳糖亚急性大鼠衰老模型已复制成功。与模型组比较,细胞治疗组血清睾酮含量有所升高(P=0.033)。
空白对照组、D-gal模型处理组、ADSCs-移植组之间的交配次数比较差异有统计学意义(F=16.280,P<0.001),与空白对照组比较,模型组大鼠交配次数有所减少(P<0.001),提示D-半乳糖亚急性大鼠衰老模型复制成功。经过移植细胞治疗后,与模型组比较,细胞治疗组交配次数有提高(P=0.027)。
睾丸间质细胞分泌3β-HSD、17β-HSD增多,而TUNEL染色显示睾丸间质细胞凋亡率降低(P<0.001)。
结论
1、筋膜结缔组织脂肪源干细胞具有长梭形、多角形的形态学特征,可以形成细胞集落,与其它组织来源间充质干细胞无显著差别；细胞表面标记物检测中,4代贴壁ADSCs的特异性表面标志物CD90.CD29表达率分别为90%以上,而CD49d、CD11b、CD45阴性表达,CD106弱表达；该细胞经定向诱导后可以成骨、成脂肪,与间充质干细胞多向分化的功能一致。由此可以判断在发育成熟大鼠的筋膜结缔组织中存在未分化间充质干细胞。
2、通过Percoll梯度离心的方法,可以获取稳定的高纯度的睾丸间质细胞；30-HSD特异性染色,可以对其鉴定。
3、中浓度的D-gal诱导细胞衰老,且可以保证细胞存活率。在高浓度环境下(16g/L)及中浓度(8g/L)的长时间作用下,可以诱使睾丸间质细胞凋亡。
4、体外脂肪干细胞基质与8g/L D-gal处理的睾丸间质细胞共培养,可以明显防护D-半乳糖对睾丸间质细胞的损害,提高细胞活性,减少衰老细胞数目。促进3p-HSD、17β-HSD的分泌,保护睾丸间质细胞。
5、连续颈背部皮下注射D-gal可以有效模拟亚急性衰老模型。衰老大鼠在外观上及自由基等方面,显示显著衰老征象。
6、尾静脉移植脂肪源干细胞后,可显著升高衰老大鼠体内SOD含量、IL-2水平,而有效降低MDA含量,同时提高胸腺和脾脏指数。
7、移植脂肪源干细胞可以改善衰老老鼠的交配能力,促进睾酮释放,同时保护睾丸间质细胞。
综上所述,本研究分别采用了细胞分离培养、诱导分化、动物模型建立、HE染色和免疫组化、免疫荧光等技术手段,从血清学自由基及免疫功能方面,以及交配能力等多方面、多角度的探讨了体内移植外源性脂肪源干细胞对D-gal诱导的亚急性衰老大鼠的作用以及体外ADSCs-CM对D-gal处理的睾丸间质细胞的保护作用。同时,体外ADSCs-CM可以降低D-gal对睾丸间质细胞的毒性作用。本研究发现,补充外源性的脂肪源干细胞可以延缓D-gal诱导的衰老,提高其生命质量；从而为筋膜学的支持与储备理论提供实验支持。
Background:
Aging is the decline of the whole body morphology and physiological function, which is a dynamic, complex process. Currently, prevention and anti-aging have become a focus of life sciences. Research on the mechanisms of aging have been the topics at the forefront of research in the field of biology, There are variety of theories explain the mechanism of geriatrics, such as the free radical theory, metabolic theory, immune system degradation doctrine, telomerase, and so on. But there is not a theory can complete explain the mechanism of aging.
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. 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. Therefore, how to maintain the normal state of fascia, continuously to provide a stable source of repair cells for the functional system and to maintain the normal function of cell division, maybe is the key to keep the human body with a longer life cycle. During the process of aging, if new cells are provided timely to the functional system, they will improve the quality and prolong the cycle of life. Unlike other theories of aging, the the fasciology from a different angle-Developmental Biology of Aging proposed a new understanding, with various theories of aging from the microscopic point of view, there is no contradiction that various microscopic point of the change. The final result is fascia tissue-support reserve system function decline.
Mesenchymal stem cells have the capability of self-renewal and multi-directional differentiation. Initially, it was found by Friendenstein, is proved that in vitro they can differentiate into osteoblast and fat cells. And then the other research team found bone marrow-derived Mesenchymal stem cells (B-MSCs) can not only differentiate into tissues from mesoderm, but also into the tissues from both endoderm and ectoderm. It is consistent from Mesenchymal stem cells'multiple sources to the fascial connective tissue's distribution, and from its biological characteristics of vertical and horizontal differentiation to the fascia connective tissue playing support and reserve role in body. ADSCs are main undifferentiated cells in the supporting-storing system. They are easy to be cultured, rich in source and low in immunity. They also have the capacity of cross-mesodermal differentiation and have endocrine functions. The potential of multi-differentiation of ADSCs makes them have developmental plasticity and makes them become a candidate of allograft. The adipose-derived stem cells can adjust the local microenvironment of the damaged tissue. Adipose-derived stem cells can secrete a series of cytokines and growth factor into the surrounding tissue fluid, to regulate tissue micro-environment, the formation of the micro-environment that is conducive to the growth of stem cells-stem cell niche, recruitment of endogenous stem cells from receptor to reach the target and to differentiated tissue cells directed to the specific needs. Adipose stem cells themselves can withstand hypoxia, can release the same time in a hypoxic environment antioxidants, free radical scavengers, molecular chaperones, heat shock proteins, etc., removal of damaged local cytotoxic substance to promote still alive cells recovery form conducive to fat grafting tissue cells to survive and function to maintain the microenvironment.
ADSC may also paracrine cytokines promote tissue repair. Intravenous injection of ADSC autonomously gathered at the lesion site involved in the repair of damage. ADSC This "homing" role, the exact mechanism is unknown. Reported that by increasing the expression of membrane-type matrix metalloproteinase1(membranetype1matrix metallop roteinase, MT1-MMP) and ADSCs promote activation of MMP-2zymogen enhances migration. Recent studies found that, ADSC release of new mitochondria to damage cells, thereby saving the process of aerobic metabolism. This shows the the ADSC is available through a variety of mechanisms involved in tissue repair. In a word, ADSCs secrete various growth factors. These proteins control and manage the damaged neighboring cells. Meanwhile, the production and secretion of growth factors has been considered as essential function of ADSCs. ADSCs also exhibit an antioxidant effect, although the mechanism is not yet clear. When ADSCs are tansplanted to the damaged animals, these stem cells may secret cytokines and growth factors to stimulate the animal to repair the damaged tissues. ADSCs have been found to produce anti-oxidants, free radical scavenger, such as heat shock protein, to promote the repair of the survival cells. ADSCs can stimulate endogenous stem cells to supplement to the target organ or can direct migrate of their own to the repair region, by which to regulate microenvironments of stem cell and promote them differentiate into functional cells, so as to replace aged functional cells to maintain the balance of the body. Further studies are necessary to understand the complete protection mechanism of ADSCs.
D-gal has been found to induce aging rats. Oversupply of D-galactose will result in abnormality of metabolism. The oxidative metabolism of D-galactose produces reactive oxygen species, which suppress the ability of the cells to eliminate them, consequently causing impairment of cellular membrane, structure, and gene expression. D-gal also can impact on cell metabolism and function of some important enzymes, which contributes to making clear the mechanism of this aging model to some extent. Its specific mechanisms of aging and functional decline may be related to metabolic disorders, immunological damage, free radical damage and mitochondrial injury related.Disorders of glucose metabolism will cause abnormal heart, liver, kidney, brain and other important organs metabolism, finally appeared senescence. D-galactose model because of the full impact of cell metabolism and some important enzyme function, caused by the aging more comprehensive. The specific mechanisms of aging and functional decline may be related to metabolic disorders, immune injury, free radical damage and mitochondrial injury related. At present, the model has been widely used in research on the mechanism of aging and delaying senility drug screening. D-gal induced subacute aging model can simulate the brain, liver, kidney, skin, gonadal axis, osteoporosis, the immune system and multiple organ damage. Reports in the literature can be caused by a variety of methods extract of Chinese medicine, Chinese medicine decoction, hormones, and acupuncture treatment of this model of aging, also reported application of stem cell transplantation in the treatment of.
Androgen can improve the excitability of the central nervous system, cause sexual impulse, thus maintaining the normal sexual function. Leydig cells (Leydig cells) is the main cell body of testosterone secretion, secretory volume accounted for about95%of total body testosterone; treatment of reproductive disorders of the male international will also focus on cultivating Leydig cells and transplantation. Leydig cell has the characteristics of synthesis, secretion of testosterone, testosterone synthesis process,-HSD317β,3β-HSD1catalytic DHEA formation of testosterone. And3β-HSD is a key enzyme in the synthesis of testosterone in adult rat testis, only mesenchymal cells expression of3β-HSD, so the current identification of mesenchymal cells generally use3β-HSD specific staining method. Leydig cell separation Percoll gradient concentrations of large, high purity, good cell vitality, in solving the problem of androgen secretion cell source for tissue seed to construct tissue engineering technology. Tissue engineering of androgen is obtained with the method of Leydig cell and the construction of the secretory tissue can maintain testosterone secretion in longer period.
The aim of this experiment is to investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms, and to verify the theory of the fasciology.In order to study the exogenous adipose-derived stem cell transplantation is a function of delaying senility and its mechanism of action, this experiment in vivo transplantation of exogenous ADSCs on D-galactose (D-gal) effects of aging rats free radicals and immune senescence index and the ability of aging rats improvement; adipose-derived stem cells in vitro matrix D-gal treatment on Leydig cells (LCs) after co-culture, exploring the protective effects of ADSCs on Leydig cell, to explore a new anti-aging mechanism, to provide new ideas and methods for clinical aging treatment.
Objective
To investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms and to verify the theory of the fasciaology.
1. To judge that whether the ADSCs exist in facial connective tissue;
2. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on free radical metabolism and immune function of rat aging model induced by D-gal from fasiaology perspective; to explore a new method for anti-aging.
3. Through mating experiments, to observe if ADSCs from fascia connective tissue can improve the aging rats induced by D-galactose sexual function; by immunohistochemical staining to location the transplanted ADSCs, labeled stem cells in the testis and displacement, and the secretion of Leydig cells of3β-HSD, 17beta-HSD. By immunohistochemical staining, morphological observation of cell changes of Leydig cells. Observation of testosterone secretion; Using Elisa to test the secretion of3β-HSD andl7p-HSD.
4. Isolation and culture of Leydig cells in vitro, to observe the protective effect of adipose-derived stem cells on D-gal treated Leydig cells.
5. Rut female rats evoke male rats to mate, to observe male rats'mate ability and mate number of times. Through radioimmunochemistry to detect testosterone content of the rats'blood serum. Observation of the expression、location and translocation of ADSCs in the testis by immunohistochemistry staining, And provide the evidence for the theory of "fasciology".
Methods
1.To detach the ADSCs from the rats'adipose tissue, to cultivate the cells in vitro and observe their formation, means of growth and biological markers on the cells'membrane, and to induce ADSCs to differentiate into adipogenic and osteogenesis, and observe their formation and specific markers. To judge whether connective tissue contains ADSCs?
2. In vitro Percoll separating medium gradient isolation and culture of Leydig cells, and identified by morphological and3β-HSD specific staining;0,4,8,12,16g/L of different concentrations of D-gal treating Leydig cells for24h, the cell survival rate was calculated, screening the best concentration simulation cell aging model; flow cytometry detection the the changes of cell cycle of8g/L D-galactose treatment Leydig cell in0,24,48,72h different time points.
3. Isolated and cultured in vitro, the4th generation ADSCs, then preparation adipose-derived stem cell matrix (ADSC-CM); while using8g/L D-galactose and adipose stem cells matrix (ADSC-CM) co-culture Leydig cells for24h, to detect the cell activity by MTT; cells were co-culture for24h, then detected by flow cytometry; co-culture of48h detection of cell senescence using SA-P-gal anhydride enzyme staining; immunohistochemical study of3β-HSD1,17β-HSD3secretion; secretion of testosterone。
4. Thirty male SD rats weighing180-200gwere randomly divided into3groups (A, B and C),30in each group. D-gal was dissolved in0.9%saline at a concentration of15%just before use.The rats in groups B and C were subcutaneously injected with D-gal at a dose of1000mg/kg body weight once every day for a total of8weeks. The rats in group A were injected with physiological saline at the same volume everyday for8weeks for experimental control.
5. Harvest and culture purified ADSCs from SD rats and these cells were labeled with Brdu for use. After all D-gal injections have been finished, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the4th passage were harvested and resuspended in the physiological saline at3×106cells/ml. Each rat in group C was injected with3x106of ADSCs into its tail vein. The rats in groups A and B were similarly administered with same volume of physiological saline.
6. Female rats were brought into behavioral estrus with sc injections of200μg/kg estradiol benzoate48h before and2mg/kg progesterone4h before testing. Only fully receptive females were used to copulate with males. The changes of structure of the testis were showed by HE stain. The changes of30-HSD expression and apototic index of leydig cells were analyzed by immunohistochemistry. The transplanted ADSCs were detect by Brdu immunohistochemistry.
7. Statistical analysis was carried out using SPSS13.0statistical software, ANOVA followed by Fisher's post hoc analysis. variance, the mean were compared with single factor analysis of variance, two-two compared with LSD method; variance not neat, multi-average number were compared by Welch method, two-two were compared with Dunnett's T3, P<0.05were significant differences.
Results
1. ADSCs can be extracted from the rats'fat tissue. In primary culture, the isolated ADSCs exhibited a fibroblast-like morphology.The two passage cells were adherent and had a spindle-shaped morphology in the third days; Flow cytometric analysis demonstrated that ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens; Culturing of undifferentiated cells for14days under adipogenic conditions induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were characteristics of adipocytes. Induction of osteogenic differentiation of the cells for14days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were characteristics of osteoblasts.
2. In vitro experiment, Leydig cells can be successfully cultured by Perpoll separation method. Leydig cells were polygon, triangle. Monolayer growth, stretching in whorls, cells in the colony form proliferation; By3β-HSD specific staining, Leydig cells are stained blue-black under microscope, and other testicular cells do not stain, Leydig cell purity can reach more than95%; MTT showed8g/L D-gal cell survival rate is the most suitable simulation model can successfully induce cell senescence, induce Leydig cells aging; with the extension of time,8g/L D-gal LCs of G0/G1phase cells were increased, S phase cells decreased.
3. ADSCs-CM was extracted with ADSCs supernatant; Leydig cells after8g/L D-gal co-culture, MTT showed improved cell activity; when co-cultured for24h, flow cytometry showed the percentage of the G0/G1phase cell decline in the cell cycle; co-culture of48h, SA-β-gal anhydride enzyme staining positive cells, co-culture galactose anhydride enzyme group SA-beta reduction, cell senescence reduced; immunohistochemistry examination showed cells secrete3β-HSD1,17β-HSD3increase.
4. In this experiment, aging model has been successfully established by chronically exposing animals to D-gal. After transplanting ADSCs, the rats in cell treatment group also showed a significant decrease in MDA activities and a significant increase in SOD levels and IL-2levels. Thymus index and spleen index also have been decreased. While the NO content have no change by contrast with the aging group.
5. We found Brdu positive cells distributed in testis tissue of experimental group SD rats after transplantion and trends to migrate to the injury area. The mate ability of aging rats have been improved by transplanting ADSCs. HE staining showed seminiferous tubules in aging group were thinner than that in normal control groups and the sperm number in the tubules decreased apparently. Immunohistochemistry showed that the level of3β-HSD expression reduced significantly compared with that in the control groups. TUNEL assay showed that ADSCs reduced apoptotic leydig cell death induced by D-gal. The testosterone contents and3β-HSD expression in cell treatment group have been significantly increased by contras with the aging group.
Conclusion
1. Fascia connective tissue derived mesenchymal stem cells have the morphological features of long spindle, and polygon, and can form cell colony. There is no significant difference with the other kinds of MSCs in morphology; ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens, in line with mesenchymal stem cell characteristics; the cells can be induced to differentiate into osteoblasts and fat cells. It can be judged that mesenchymal stem cells exist in mature rats'facial connective tissue.
2. Through the method of Percoll gradient centrifugation, can obtain the high purity of Leydig cells; Leydig cells can be identified by3β-HSD specific staining,.
3. The concentration of D-gal can ensure the survival rate of cells, induce cell senescence. Under high concentration environment (16g/L) and concentration (8g/L) of the long time effect, can induce apoptosis in Leydig cells.
4. ADSCs-CM and8g/L D-gal treatment of Leydig cells, ADSCs-CM can effectively protect Leydig cell from D-galactose damage; ADSCs can increase cell activity, reduce the number of cell senescence. Promote the secretion of3β-HSD,17P-HSD, protect Leydig cells.
5. Rodents injected with D-gal for6-10weeks can successfully establish Sub-acute aging model, which shows progressive deterioration of learning and memory capacity and mate ability, increased production of free radicals in the body.
6. Through transplanting ADSCs to the aging rats, SOD levels、IL-2levels、 thymus index and spleen index have a significant increase in cell treatment group rats while contrast with the aging model rats. While MDA activities show a significant decreased.
7. Transplanting ADSCs can improve the mate ability of aging rats, stimulate the increase of the testosterone contents and3β-HSD expression, improve the quality of life.
In summary, the research initiated to study the effects of transplanting ADSCs to D-gal induced aging rats on free radical metabolism、immune function、the capability of spatial learning and memory and mate ability of rats aging model induced by D-gal from fasiology perspective, through cell culture, HE staining, immunhistochemistry technology.That to explore the mechanism of transplanting ADSCs to aging rats is a new try of anti-aging therapies in the point of fasiology. In this experiment,supplementing the D-gal induced aging rats with exogenous stem cells can delay the aging procedure, improve the quality of the life.
衰老(senescence)又称老化,是整个机体形态结构和生理机能的全面衰退,是一个动态的、复杂的过程。当前预防和延缓衰老已成为生命科学研究的热点。有关衰老机制的研究一直是生物学、老年医学研究的前沿课题。对其机制的研究中有自由基学说、代谢学说、免疫系统退化学说、端粒酶变化等多种学说,但没有一种学说能完整解释衰老的机制。
原林教授提出的“筋膜学说”认为：人体由两部分组成,一个是由尚未分化的非特异性结缔组织(筋膜)支架所构成的支持与储备系统,一个是以已分化的功能细胞为基础的功能系统。功能系统在支持与储备系统支持下维持结构与功能的稳定,而支持与储备系统源源不断的为功能系统的细胞更新和功能活动提供细胞供应和营养物质。功能系统是以功能细胞的专能特化为基础的,而支持与储备系统的筋膜支架以干细胞为核心,通过分化出各种定向干细胞,为功能系统的更新提供源源不断的细胞补充,并为功能系统的各种细胞的更新、代谢提供一个稳定的内部环境。即衰老是筋膜中干细胞储备逐渐耗竭的过程,而如何保持筋膜的正常状态,从而为功能系统提供稳定的细胞来源并维持其向功能细胞的正常分化,是延长生命周期的关键。如果衰老时减少的干细胞能得到及时的补充,为机体提供新的细胞储备,可修复机体损伤,延缓衰老、延长生命周期。不同于其他的衰老学说,筋膜学说从另一个不同的角度——发育生物学——对衰老提出了新的认识,同时与从微观角度提出的各种衰老学说并无矛盾,认为各种微观角度的改变最终导致的结果都是筋膜组织—支持与储备系统功能的减退。
间充质干细胞(menchymal stem cells)具有自我更新、多向分化的能力。最初是由Friendenstein等发现,而且证明其在体外可以分化成为成骨细胞及脂肪细胞,而后其它的研究小组发现骨髓来源的MSCs不仅可以分化为中胚层来源的组织,而且可以分化为内胚层和外胚层来源的组织。而且间充质干细胞的多组织来源与筋膜结缔组织支架的全身分布是一致的,其纵向、横向分化的生物学特性与筋膜学提出的筋膜结缔组织对机体的支持储备作用也是一致的。脂肪源干细胞(ADSCs)是筋膜中未分化干细胞的一种储备形式。ADSCs具有多向分化潜能,能够跨胚层分化。它可以分化成中胚层来源的脂肪细胞、成骨细胞、软骨细胞、心肌细胞；外胚层来源的神经细胞；也可以分化成内胚层来源的内皮细胞及肝细胞等。ADSCs具有低免疫原性,免疫调节功能和分泌细胞因子等能力,为异体移植提供了有利条件,使ADSCs成为生物治疗的亮点之一；ADSCs分泌细胞因子抗老化的作用,已经被临床广泛应用。ADSCs不仅可以用来进行各种退行性和衰竭疑难病的替代治疗,而且可用来进行辅助的免疫治疗。脂肪源干细胞可以调节受损组织局部微环境,可以分泌一系列细胞因子和生长因子到周围组织液中,调节组织微环境,形成有利于干细胞生长的微环境--“干细胞巢”--招募来自于受体的内源性干细胞到达靶点,并使其向所需要的特定组织细胞定向分化。脂肪干细胞本身能够耐受缺氧,同时在缺氧的环境中还能释放抗氧化物质、自由基清除剂、分子伴侣、热休克蛋白等,清除受损局部的细胞毒性物质,促进尚存活的细胞复苏,在脂肪移植后形成有利于组织细胞存活和功能维持的微环境。ADSC还有可能通过旁分泌细胞因子促进组织修复。经静脉注射的ADSC可自主聚集于病灶部位参与修复损伤。ADSC的这种“归巢”作用,其具体机制不明。有报道称通过增加膜型基质金属蛋白酶1(membranetype1matrix metallop roteinase, MT1-MMP)表达和促进MMP-2酶原的激活可增强ADSCs的迁移。近期研究发现,ADSC能释放新的线粒体至损害的细胞,从而挽救有氧代谢的过程。这说明ADSC可通过多种机制参与组织的修复。
简言之,ADSCs可能通过以下机制修复或者重建组织：首先,ADSCs被植入一个受损的或是坏死的组织,它们能以副分泌的方式分泌出细胞活素和生长因子以刺激修复。ADSCs可以提供抗氧化剂、自由基清除剂、热休克蛋白等给待修复组织,从而清除局部环境中所释放的有毒物质,促进仍存活的细胞的修复。另外,ADSCs可以通过刺激内生性干细胞补充到待修复的脏器或自身直接迁移补充到待修复区域,以调节干细胞的微环境,并且促进它们沿着需要的方式分化为功能细胞,从而更新机体衰老的功能细胞,维持机体平衡。
D-gal所致亚急性衰老模型是国内外比较公认的衰老模型。在一定时间内连续给动物注射D-半乳糖使机体细胞糖代谢及脂代谢紊乱,体内糖浓度增高后被醛糖还原酶催化还原成半乳糖醇,其不能被细胞进一步代谢而堆积体内、影响正常渗透压,导致细胞肿胀、功能障碍、代谢紊乱,不仅破坏并消耗机体抗氧化防御系统,使自由基积聚；同时在半乳糖还原成半乳糖醇的过程中又使细胞膜脂质受损,过氧化脂质、脂褐素增高,出现衰老。糖代谢紊乱必然会引起心、肝、肾、脑等重要器官代谢异常,最后出现衰老。D-半乳糖模型因全面影响细胞的代谢功能和一些重要的酶的功能,所导致的老化较全面。其老化与机能减退的具体机制可能与代谢障碍、免疫损伤、自由基损伤及线粒体损伤等相关。目前,该模型已广泛应用于研究衰老机制以及延缓衰老药物的筛选。D-gal致亚急性衰老模型可以模拟脑、肝脏、肾脏、皮肤、性腺轴、骨质疏松、免疫系统等多脏器的损伤。文献报道可应用干细胞移植治疗,亦有文献报道应用中药提取物、中药汤方、激素及针灸等多种方法治疗此模型造成的衰老。
雄激素可提高性中枢神经系统的兴奋性,诱发性欲冲动,从而维持正常的性机能。睾丸间质细胞(Leydig cells)是机体分泌睾酮的主要细胞,分泌量约占身体总睾酮的95%；国际上对男性生殖疾病治疗的研究也多集中在Leydig细胞的培养和移植上。睾丸间质细胞具有合成、分泌睾酮的特点。睾酮合成过程中,17β-HSD3、3p-HSD1催化脱氢异雄酮形成睾酮。而3β-HSD是合成睾酮的关键酶,成年大鼠睾丸中只有间质细胞表达3β-HSD,所以目前对间质细胞的鉴定一般都采用3β-HSD特异性染色方法。Percoll梯度浓度分离的Leydig细胞量大、纯度较高、细胞活力良好,初步解决了组织工程技术构建雄激素分泌组织的种子细胞来源的问题。用该方法获得的Leydig细胞所构建的组织工程化雄激素分泌组织能在较长时期维持睾酮分泌功能。
为探讨外源性脂肪源干细胞移植是否具有延缓衰老的作用及其作用机制,本实验体内观察移植外源性ADSCs对D-半乳糖(D-gal)衰老大鼠自由基及免疫等衰老指标,以及对衰老大鼠性能力的改善等方面的影响；体外观察脂肪源干细胞基质与D-gal处理的睾丸间质细胞(LCs)共培养,探讨ADSCs对Leydig cell的保护性作用,探索一种新的抗衰老机理,为临床延缓衰老的治疗提供新的思路和方法。
目的
研究移植脂肪源干细胞对D-半乳糖致衰老大鼠相关衰老指标以及交配能力的影响,探讨临床替代疗法的治疗机理,进一步为筋膜结缔组织是干细胞的储备源泉提供证据支持,为筋膜学提供理论支撑。
1、检验非特异性筋膜结缔组织中是否存在脂肪源干细胞。
2、观察移植脂肪源干细胞能否对D-半乳糖衰老大鼠的血清、自由基及免疫功能产生影响,阐述移植脂肪源干细胞对抗D-半乳糖衰老的血清学意义。
3、通过观察交配实验,研究移植筋膜结缔组织中的脂肪源干细胞能否提高D-半乳糖致衰老大鼠的性能力；通过免疫组织化学染色,观察移植标记的脂肪源干细胞在睾丸组织中的定位和移位情况,及睾丸间质细胞分泌3p-HSD、17β-HSD的情况；通过免疫组织化学染色,观察睾丸间质细胞细胞形态学变化及其凋亡状况；通过血清学检测,观察睾酮分泌状况。
4、体外分离培养睾丸间质细胞,观察脂肪干细胞对D-gal处理的睾丸间质细胞的保护性作用,为“筋膜学”抗衰老相关理论提供进一步佐证。
方法
1、取成年SD大鼠腹股沟脂肪垫,用酶消化法分离、培养筋膜结缔组织的脂肪源干细胞。通过用形态学、功能学、流式细胞术鉴定非特异性筋膜结缔组织源细胞是否符合间充质干细胞的特性,进而判断非特异性筋膜结缔组织中是否含有间充质干细胞。
2、体外Percoll分离液梯度分离培养睾丸间质细胞,并通过形态学及3β-HSD特异性染色鉴定；以0、4、8、12、16g/L不同浓度的D-gal处理睾丸间质细胞,每个浓度做三个复孔,每个孔重复三次。24h后,计算细胞存活率,筛选最适浓度模拟细胞衰老模型；另流式细胞仪检测8g/LD-半乳糖处理Leydig cell在0、24、48、72h不同时间点的细胞周期变化。
3、体外分离培养4代ADSCs,取上清制备脂肪干细胞基质(ADSC-CM);8g/LD-半乳糖处理Leydig cell,同时与ADSC-CM共培养24h后,MTT检测其细胞活性；流式细胞仪检测各组细胞共培养24h的周期变化；共培养48h,SA-p-半乳糖酐酶染色,每组取4个视野,每个视野至少100个细胞。检测细胞衰老情况；免疫组化观察3p-HSD分泌情况。
4、30只SD大鼠随机分成空白对照组(A组)、模型组(B组)和治疗组(C组),每组10只。B、C组颈背部皮下注射15%D-半乳糖1000mg/(kg-d),连续8周复制亚急性衰老模型,A组的大鼠每天给予生理盐水1.6ml颈背部皮下注射。
5、模拟衰老模型成功后,治疗组内的大鼠进行脂肪源干细胞静脉移植：移植的第3代ADSCs在进行传代后,在培养基中掺入10μmol/L的Brdu,孵育3天后消化收集,无菌生理盐水重悬,制成300万/ml；同样每只大鼠经尾静脉注射ADSCs3×106个,模型组输入生理盐水1ml。
6、交配实验的雌鼠分别在实验前48h和4h采用皮下注射苯甲酸雌二醇200μg/kg和黄体酮2mg/kg用来诱导雌鼠发情,用发情的雌鼠和雄鼠进行交配,从而对雄性进行相关性能力研究。HE染色显示大鼠睾丸组织精曲小管结构变化,采用免疫组化及免疫荧光追踪移植细胞；免疫组化观察3β-HSD1、17β-HSD3分泌情况,TUNEL免疫组化染色观察大鼠间质细胞凋亡指数。放射免疫法观察大鼠血清睾酮含量。
7、采用SPSS13.0统计软件处理,以均数±标准差(x±s)表示,方差齐时,多样本均数比较均采用单因素方差分析,两两比较采用LSD法；方差不齐时,多样本均数比较均采用Welch法,两两比较采用Dunnett's T3方法,P<0.05表示有统计学差异。
结果
1、腹股沟皮下筋膜结缔组织内的脂肪源干细胞具有长梭形、多角形的形态学特征,可形成细胞集落,体外可以诱导分化为成骨细胞、脂肪细胞。
2、体外Percoll分离法可成功分离培养睾丸间质细胞。间质细胞呈多边形、三角形。单层贴壁生长,伸展后呈漩涡状排列,细胞以集落形式增殖；经3p-HSD特异性染色鉴定,镜下可见睾丸间质细胞被染成蓝黑色而其他睾丸细胞不着色,睾丸间质细胞纯度可达到95%以上；MTT法显示8g/L的D-gal模拟衰老模型细胞存活率为(81.96±0.96),为最适浓度,可成功诱导leydig cell老化(F=8960.772,P=0.000)；且随时间延长,8g/L的D-gal处理的LCs呈现G0/G1期细胞增多,S期细胞减少。
3、ADSCs上清中可以提取出ADSCs-CM。空白对照组、8g/LD-gal处理的Leydig cells组、8g/LD-gal处理的Leydig cells与ADSCs-CM共培养组做比较,三组之间差异有统计学意义(F=1101.980,P<0.001)。MTT检测显示其细胞活性有所提高(P<0.001)；流式细胞仪共培养24h后,细胞周期中G0/G1期细胞比例下降；
空白对照组、8g/LD-gal处理的Leydig cells组、共培养组之间的SA-β-半乳糖酐酶阳性细胞数差异有统计学意义(F=1105.307,P<0.001)；共培养48h, SA-β-半乳糖酐酶染色,共培养组SA-β-半乳糖酐酶阳性细胞减少(P<0.001),细胞衰老减轻；免疫组化观察共培养后细胞分泌的3β-HSD分泌情况增加。
4、连续皮下注射D-gal可以模拟大鼠衰老。空白对照组、D-gal处理组、ADSCs-移植组之间的CuZn-SOD (F=28.587, P<0.001)、NO水平(F=56.571,P<0.001)、血清IL-2水平(F=23.667,P<0.001)、脾脏指数(F=33.830,P<0.001)和胸腺指数(F=15.847, P<0.001)、MDA水平(F=45.468,P<0.001)差异有统计学意义。衰老模型组与空白对照组比较,血清MDA水平有所升高(P<0.001),血清CuZn-SOD(P<0.001)、血清NO(P<0.001、IL-2水平(P<0.001)以及脾脏指数(P<0.001)、胸腺指数(P<0.001)均有所降低,提示D-半乳糖模拟亚急性大鼠衰老模型复制成功。与模型组比较,细胞治疗后衰老大鼠SOD活性(P=0.04)、血清IL-2水平(p=0.038)、血清NO水平(P<0.001)和胸腺指数(P=0.002)、脾脏指数(P=0.001)均有所升高,MDA水平则降低(P<0.001)。
5、空白对照组、D-gal模型处理组、ADSCs-移植组之间的血清睾酮含量比较差异有统计学意义(F=25.370,P<0.001),与空白对照组比较,模型组血清睾酮含量有所降低(P<0.001),提示D-半乳糖亚急性大鼠衰老模型已复制成功。与模型组比较,细胞治疗组血清睾酮含量有所升高(P=0.033)。
空白对照组、D-gal模型处理组、ADSCs-移植组之间的交配次数比较差异有统计学意义(F=16.280,P<0.001),与空白对照组比较,模型组大鼠交配次数有所减少(P<0.001),提示D-半乳糖亚急性大鼠衰老模型复制成功。经过移植细胞治疗后,与模型组比较,细胞治疗组交配次数有提高(P=0.027)。
睾丸间质细胞分泌3β-HSD、17β-HSD增多,而TUNEL染色显示睾丸间质细胞凋亡率降低(P<0.001)。
结论
1、筋膜结缔组织脂肪源干细胞具有长梭形、多角形的形态学特征,可以形成细胞集落,与其它组织来源间充质干细胞无显著差别；细胞表面标记物检测中,4代贴壁ADSCs的特异性表面标志物CD90.CD29表达率分别为90%以上,而CD49d、CD11b、CD45阴性表达,CD106弱表达；该细胞经定向诱导后可以成骨、成脂肪,与间充质干细胞多向分化的功能一致。由此可以判断在发育成熟大鼠的筋膜结缔组织中存在未分化间充质干细胞。
2、通过Percoll梯度离心的方法,可以获取稳定的高纯度的睾丸间质细胞；30-HSD特异性染色,可以对其鉴定。
3、中浓度的D-gal诱导细胞衰老,且可以保证细胞存活率。在高浓度环境下(16g/L)及中浓度(8g/L)的长时间作用下,可以诱使睾丸间质细胞凋亡。
4、体外脂肪干细胞基质与8g/L D-gal处理的睾丸间质细胞共培养,可以明显防护D-半乳糖对睾丸间质细胞的损害,提高细胞活性,减少衰老细胞数目。促进3p-HSD、17β-HSD的分泌,保护睾丸间质细胞。
5、连续颈背部皮下注射D-gal可以有效模拟亚急性衰老模型。衰老大鼠在外观上及自由基等方面,显示显著衰老征象。
6、尾静脉移植脂肪源干细胞后,可显著升高衰老大鼠体内SOD含量、IL-2水平,而有效降低MDA含量,同时提高胸腺和脾脏指数。
7、移植脂肪源干细胞可以改善衰老老鼠的交配能力,促进睾酮释放,同时保护睾丸间质细胞。
综上所述,本研究分别采用了细胞分离培养、诱导分化、动物模型建立、HE染色和免疫组化、免疫荧光等技术手段,从血清学自由基及免疫功能方面,以及交配能力等多方面、多角度的探讨了体内移植外源性脂肪源干细胞对D-gal诱导的亚急性衰老大鼠的作用以及体外ADSCs-CM对D-gal处理的睾丸间质细胞的保护作用。同时,体外ADSCs-CM可以降低D-gal对睾丸间质细胞的毒性作用。本研究发现,补充外源性的脂肪源干细胞可以延缓D-gal诱导的衰老,提高其生命质量；从而为筋膜学的支持与储备理论提供实验支持。
Background:
Aging is the decline of the whole body morphology and physiological function, which is a dynamic, complex process. Currently, prevention and anti-aging have become a focus of life sciences. Research on the mechanisms of aging have been the topics at the forefront of research in the field of biology, There are variety of theories explain the mechanism of geriatrics, such as the free radical theory, metabolic theory, immune system degradation doctrine, telomerase, and so on. But there is not a theory can complete explain the mechanism of aging.
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. 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. Therefore, how to maintain the normal state of fascia, continuously to provide a stable source of repair cells for the functional system and to maintain the normal function of cell division, maybe is the key to keep the human body with a longer life cycle. During the process of aging, if new cells are provided timely to the functional system, they will improve the quality and prolong the cycle of life. Unlike other theories of aging, the the fasciology from a different angle-Developmental Biology of Aging proposed a new understanding, with various theories of aging from the microscopic point of view, there is no contradiction that various microscopic point of the change. The final result is fascia tissue-support reserve system function decline.
Mesenchymal stem cells have the capability of self-renewal and multi-directional differentiation. Initially, it was found by Friendenstein, is proved that in vitro they can differentiate into osteoblast and fat cells. And then the other research team found bone marrow-derived Mesenchymal stem cells (B-MSCs) can not only differentiate into tissues from mesoderm, but also into the tissues from both endoderm and ectoderm. It is consistent from Mesenchymal stem cells'multiple sources to the fascial connective tissue's distribution, and from its biological characteristics of vertical and horizontal differentiation to the fascia connective tissue playing support and reserve role in body. ADSCs are main undifferentiated cells in the supporting-storing system. They are easy to be cultured, rich in source and low in immunity. They also have the capacity of cross-mesodermal differentiation and have endocrine functions. The potential of multi-differentiation of ADSCs makes them have developmental plasticity and makes them become a candidate of allograft. The adipose-derived stem cells can adjust the local microenvironment of the damaged tissue. Adipose-derived stem cells can secrete a series of cytokines and growth factor into the surrounding tissue fluid, to regulate tissue micro-environment, the formation of the micro-environment that is conducive to the growth of stem cells-stem cell niche, recruitment of endogenous stem cells from receptor to reach the target and to differentiated tissue cells directed to the specific needs. Adipose stem cells themselves can withstand hypoxia, can release the same time in a hypoxic environment antioxidants, free radical scavengers, molecular chaperones, heat shock proteins, etc., removal of damaged local cytotoxic substance to promote still alive cells recovery form conducive to fat grafting tissue cells to survive and function to maintain the microenvironment.
ADSC may also paracrine cytokines promote tissue repair. Intravenous injection of ADSC autonomously gathered at the lesion site involved in the repair of damage. ADSC This "homing" role, the exact mechanism is unknown. Reported that by increasing the expression of membrane-type matrix metalloproteinase1(membranetype1matrix metallop roteinase, MT1-MMP) and ADSCs promote activation of MMP-2zymogen enhances migration. Recent studies found that, ADSC release of new mitochondria to damage cells, thereby saving the process of aerobic metabolism. This shows the the ADSC is available through a variety of mechanisms involved in tissue repair. In a word, ADSCs secrete various growth factors. These proteins control and manage the damaged neighboring cells. Meanwhile, the production and secretion of growth factors has been considered as essential function of ADSCs. ADSCs also exhibit an antioxidant effect, although the mechanism is not yet clear. When ADSCs are tansplanted to the damaged animals, these stem cells may secret cytokines and growth factors to stimulate the animal to repair the damaged tissues. ADSCs have been found to produce anti-oxidants, free radical scavenger, such as heat shock protein, to promote the repair of the survival cells. ADSCs can stimulate endogenous stem cells to supplement to the target organ or can direct migrate of their own to the repair region, by which to regulate microenvironments of stem cell and promote them differentiate into functional cells, so as to replace aged functional cells to maintain the balance of the body. Further studies are necessary to understand the complete protection mechanism of ADSCs.
D-gal has been found to induce aging rats. Oversupply of D-galactose will result in abnormality of metabolism. The oxidative metabolism of D-galactose produces reactive oxygen species, which suppress the ability of the cells to eliminate them, consequently causing impairment of cellular membrane, structure, and gene expression. D-gal also can impact on cell metabolism and function of some important enzymes, which contributes to making clear the mechanism of this aging model to some extent. Its specific mechanisms of aging and functional decline may be related to metabolic disorders, immunological damage, free radical damage and mitochondrial injury related.Disorders of glucose metabolism will cause abnormal heart, liver, kidney, brain and other important organs metabolism, finally appeared senescence. D-galactose model because of the full impact of cell metabolism and some important enzyme function, caused by the aging more comprehensive. The specific mechanisms of aging and functional decline may be related to metabolic disorders, immune injury, free radical damage and mitochondrial injury related. At present, the model has been widely used in research on the mechanism of aging and delaying senility drug screening. D-gal induced subacute aging model can simulate the brain, liver, kidney, skin, gonadal axis, osteoporosis, the immune system and multiple organ damage. Reports in the literature can be caused by a variety of methods extract of Chinese medicine, Chinese medicine decoction, hormones, and acupuncture treatment of this model of aging, also reported application of stem cell transplantation in the treatment of.
Androgen can improve the excitability of the central nervous system, cause sexual impulse, thus maintaining the normal sexual function. Leydig cells (Leydig cells) is the main cell body of testosterone secretion, secretory volume accounted for about95%of total body testosterone; treatment of reproductive disorders of the male international will also focus on cultivating Leydig cells and transplantation. Leydig cell has the characteristics of synthesis, secretion of testosterone, testosterone synthesis process,-HSD317β,3β-HSD1catalytic DHEA formation of testosterone. And3β-HSD is a key enzyme in the synthesis of testosterone in adult rat testis, only mesenchymal cells expression of3β-HSD, so the current identification of mesenchymal cells generally use3β-HSD specific staining method. Leydig cell separation Percoll gradient concentrations of large, high purity, good cell vitality, in solving the problem of androgen secretion cell source for tissue seed to construct tissue engineering technology. Tissue engineering of androgen is obtained with the method of Leydig cell and the construction of the secretory tissue can maintain testosterone secretion in longer period.
The aim of this experiment is to investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms, and to verify the theory of the fasciology.In order to study the exogenous adipose-derived stem cell transplantation is a function of delaying senility and its mechanism of action, this experiment in vivo transplantation of exogenous ADSCs on D-galactose (D-gal) effects of aging rats free radicals and immune senescence index and the ability of aging rats improvement; adipose-derived stem cells in vitro matrix D-gal treatment on Leydig cells (LCs) after co-culture, exploring the protective effects of ADSCs on Leydig cell, to explore a new anti-aging mechanism, to provide new ideas and methods for clinical aging treatment.
Objective
To investigate the effects of transplanted ADSCs on D-gal induced aging rats and the possible mechanisms and to verify the theory of the fasciaology.
1. To judge that whether the ADSCs exist in facial connective tissue;
2. To observe the effect of transplanting the adipose-deived stem cells (ADSCs) on free radical metabolism and immune function of rat aging model induced by D-gal from fasiaology perspective; to explore a new method for anti-aging.
3. Through mating experiments, to observe if ADSCs from fascia connective tissue can improve the aging rats induced by D-galactose sexual function; by immunohistochemical staining to location the transplanted ADSCs, labeled stem cells in the testis and displacement, and the secretion of Leydig cells of3β-HSD, 17beta-HSD. By immunohistochemical staining, morphological observation of cell changes of Leydig cells. Observation of testosterone secretion; Using Elisa to test the secretion of3β-HSD andl7p-HSD.
4. Isolation and culture of Leydig cells in vitro, to observe the protective effect of adipose-derived stem cells on D-gal treated Leydig cells.
5. Rut female rats evoke male rats to mate, to observe male rats'mate ability and mate number of times. Through radioimmunochemistry to detect testosterone content of the rats'blood serum. Observation of the expression、location and translocation of ADSCs in the testis by immunohistochemistry staining, And provide the evidence for the theory of "fasciology".
Methods
1.To detach the ADSCs from the rats'adipose tissue, to cultivate the cells in vitro and observe their formation, means of growth and biological markers on the cells'membrane, and to induce ADSCs to differentiate into adipogenic and osteogenesis, and observe their formation and specific markers. To judge whether connective tissue contains ADSCs?
2. In vitro Percoll separating medium gradient isolation and culture of Leydig cells, and identified by morphological and3β-HSD specific staining;0,4,8,12,16g/L of different concentrations of D-gal treating Leydig cells for24h, the cell survival rate was calculated, screening the best concentration simulation cell aging model; flow cytometry detection the the changes of cell cycle of8g/L D-galactose treatment Leydig cell in0,24,48,72h different time points.
3. Isolated and cultured in vitro, the4th generation ADSCs, then preparation adipose-derived stem cell matrix (ADSC-CM); while using8g/L D-galactose and adipose stem cells matrix (ADSC-CM) co-culture Leydig cells for24h, to detect the cell activity by MTT; cells were co-culture for24h, then detected by flow cytometry; co-culture of48h detection of cell senescence using SA-P-gal anhydride enzyme staining; immunohistochemical study of3β-HSD1,17β-HSD3secretion; secretion of testosterone。
4. Thirty male SD rats weighing180-200gwere randomly divided into3groups (A, B and C),30in each group. D-gal was dissolved in0.9%saline at a concentration of15%just before use.The rats in groups B and C were subcutaneously injected with D-gal at a dose of1000mg/kg body weight once every day for a total of8weeks. The rats in group A were injected with physiological saline at the same volume everyday for8weeks for experimental control.
5. Harvest and culture purified ADSCs from SD rats and these cells were labeled with Brdu for use. After all D-gal injections have been finished, all rats in group C were injected with ADSCs. In brief, the purified ADSCs of the4th passage were harvested and resuspended in the physiological saline at3×106cells/ml. Each rat in group C was injected with3x106of ADSCs into its tail vein. The rats in groups A and B were similarly administered with same volume of physiological saline.
6. Female rats were brought into behavioral estrus with sc injections of200μg/kg estradiol benzoate48h before and2mg/kg progesterone4h before testing. Only fully receptive females were used to copulate with males. The changes of structure of the testis were showed by HE stain. The changes of30-HSD expression and apototic index of leydig cells were analyzed by immunohistochemistry. The transplanted ADSCs were detect by Brdu immunohistochemistry.
7. Statistical analysis was carried out using SPSS13.0statistical software, ANOVA followed by Fisher's post hoc analysis. variance, the mean were compared with single factor analysis of variance, two-two compared with LSD method; variance not neat, multi-average number were compared by Welch method, two-two were compared with Dunnett's T3, P<0.05were significant differences.
Results
1. ADSCs can be extracted from the rats'fat tissue. In primary culture, the isolated ADSCs exhibited a fibroblast-like morphology.The two passage cells were adherent and had a spindle-shaped morphology in the third days; Flow cytometric analysis demonstrated that ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens; Culturing of undifferentiated cells for14days under adipogenic conditions induced the formation of lipid-filled vesicles that were stained red by oil-red-O staining and were characteristics of adipocytes. Induction of osteogenic differentiation of the cells for14days resulted in the deposition of mineralized nodules that were stained red by Alizarin red staining and were characteristics of osteoblasts.
2. In vitro experiment, Leydig cells can be successfully cultured by Perpoll separation method. Leydig cells were polygon, triangle. Monolayer growth, stretching in whorls, cells in the colony form proliferation; By3β-HSD specific staining, Leydig cells are stained blue-black under microscope, and other testicular cells do not stain, Leydig cell purity can reach more than95%; MTT showed8g/L D-gal cell survival rate is the most suitable simulation model can successfully induce cell senescence, induce Leydig cells aging; with the extension of time,8g/L D-gal LCs of G0/G1phase cells were increased, S phase cells decreased.
3. ADSCs-CM was extracted with ADSCs supernatant; Leydig cells after8g/L D-gal co-culture, MTT showed improved cell activity; when co-cultured for24h, flow cytometry showed the percentage of the G0/G1phase cell decline in the cell cycle; co-culture of48h, SA-β-gal anhydride enzyme staining positive cells, co-culture galactose anhydride enzyme group SA-beta reduction, cell senescence reduced; immunohistochemistry examination showed cells secrete3β-HSD1,17β-HSD3increase.
4. In this experiment, aging model has been successfully established by chronically exposing animals to D-gal. After transplanting ADSCs, the rats in cell treatment group also showed a significant decrease in MDA activities and a significant increase in SOD levels and IL-2levels. Thymus index and spleen index also have been decreased. While the NO content have no change by contrast with the aging group.
5. We found Brdu positive cells distributed in testis tissue of experimental group SD rats after transplantion and trends to migrate to the injury area. The mate ability of aging rats have been improved by transplanting ADSCs. HE staining showed seminiferous tubules in aging group were thinner than that in normal control groups and the sperm number in the tubules decreased apparently. Immunohistochemistry showed that the level of3β-HSD expression reduced significantly compared with that in the control groups. TUNEL assay showed that ADSCs reduced apoptotic leydig cell death induced by D-gal. The testosterone contents and3β-HSD expression in cell treatment group have been significantly increased by contras with the aging group.
Conclusion
1. Fascia connective tissue derived mesenchymal stem cells have the morphological features of long spindle, and polygon, and can form cell colony. There is no significant difference with the other kinds of MSCs in morphology; ADSCs were uniformly expressed CD29, CD90, and low CD106, and negative for CD49d, CD11b and CD45surface antigens, in line with mesenchymal stem cell characteristics; the cells can be induced to differentiate into osteoblasts and fat cells. It can be judged that mesenchymal stem cells exist in mature rats'facial connective tissue.
2. Through the method of Percoll gradient centrifugation, can obtain the high purity of Leydig cells; Leydig cells can be identified by3β-HSD specific staining,.
3. The concentration of D-gal can ensure the survival rate of cells, induce cell senescence. Under high concentration environment (16g/L) and concentration (8g/L) of the long time effect, can induce apoptosis in Leydig cells.
4. ADSCs-CM and8g/L D-gal treatment of Leydig cells, ADSCs-CM can effectively protect Leydig cell from D-galactose damage; ADSCs can increase cell activity, reduce the number of cell senescence. Promote the secretion of3β-HSD,17P-HSD, protect Leydig cells.
5. Rodents injected with D-gal for6-10weeks can successfully establish Sub-acute aging model, which shows progressive deterioration of learning and memory capacity and mate ability, increased production of free radicals in the body.
6. Through transplanting ADSCs to the aging rats, SOD levels、IL-2levels、 thymus index and spleen index have a significant increase in cell treatment group rats while contrast with the aging model rats. While MDA activities show a significant decreased.
7. Transplanting ADSCs can improve the mate ability of aging rats, stimulate the increase of the testosterone contents and3β-HSD expression, improve the quality of life.
In summary, the research initiated to study the effects of transplanting ADSCs to D-gal induced aging rats on free radical metabolism、immune function、the capability of spatial learning and memory and mate ability of rats aging model induced by D-gal from fasiology perspective, through cell culture, HE staining, immunhistochemistry technology.That to explore the mechanism of transplanting ADSCs to aging rats is a new try of anti-aging therapies in the point of fasiology. In this experiment,supplementing the D-gal induced aging rats with exogenous stem cells can delay the aging procedure, improve the quality of the life.
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