微囊化人降钙素基因修饰细胞治疗绝经后骨质疏松症的研究
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摘要
背景:原发性骨质疏松症为一种以骨量减少,骨微结构破坏,导致骨脆性增加,容易发生骨折为特征的全身代谢性骨病。绝经后骨质疏松症是最常见的一种原发性骨质疏松,该病的致残率及致死率较高,是严重威胁老年人生存质量的多发病,由此产生的医疗费用成为家庭和社会的沉重负担。绝经后骨质疏松症其特征是多在绝经后5～10年内发病,主要由绝经后雌激素减少引起。雌激素水平低下时骨转换速度加快。在骨重建高转换状态下,破骨细胞骨吸收形成的陷窝得不到充分的骨形成修复,骨小梁可穿孔、断裂,不连接的小梁数增多,是导致绝经后骨量大量流失和骨质量降低的主要病理机制。绝经后骨代谢为高转换型,骨吸收大于骨形成,因而发生骨量丢失。应用骨吸收抑制剂是合理的用药选择。骨吸收抑制剂主要有雌激素类、选择性雌激素受体调节剂、双膦酸盐类及降钙素类等。其中降钙素主要由哺乳动物甲状腺旁滤泡细胞(C细胞)分泌的多肽类激素,由32个氨基酸组成。降钙素对骨的作用是抑制破骨细胞的活性和增生,从而抑制骨吸收,降低骨转换率。因此降钙素作为抗骨吸收药物在骨质疏松症临床治疗中得以广泛应用。尽管降钙素在治疗骨代谢疾病方面效果良好,但是降钙素与绝经后骨量丢失之间的关系,尚存在争议。降钙素在体内的半衰期短,要达临床满意效果,必须长期反复应用。这使很多病人难以坚持。而且此类药物有一定的抗原性,限制其临床应用。
目的:本研究首先拟观察去势大鼠基础值及其储备功能的改变;同时了解体内甲状腺C细胞形态变化特点。为进一步探讨降钙素在绝经后骨质疏松症发生中的作用提供试验依据。同时,拟微囊技术和转基因技术结合起来,通过脂质体转染方法建立降钙素基因高表达成肌细胞株,再将其微囊化,形成微囊化转降钙素基因修饰细胞。观察重组人降钙素对体外骨重建功能细胞的影响。进而将微囊化转基因修饰细胞移植体内,观察对去势大鼠的骨代谢与骨结构的影响。
方法:6月龄清洁级健康雌性SD大鼠16只,随机均分为去卵巢组和假手术组。去势12周后用钙负荷-降钙素兴奋试验分别观察两组降钙素基础值及其储备功能的改变。并处死每组大鼠,留取甲状腺组织,采用免疫组织化学方法显示降钙素阳性细胞并对其进行细胞形态计量学分析。另外,利用脂质体介导法将人降钙素基因(hCT)转染成肌细胞,以G418筛选出阳性克隆并扩大培养。用逆转录-聚合酶链反应(RT-PCR)、Western Blot和免疫组织化学分析目的基因及其蛋白表达;并用ELISA法检测上清中分泌的人降钙素浓度。将转染有hCT cDNA的L6成肌细胞包被在具有免疫隔离作用的海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊内形成微囊化基因转染细胞。在体外培养的同时,应用原代培养成骨细胞和破骨细胞方法,观察重组人降钙素对细胞形态、功能的影响。而后将微囊化基因转染细胞移植到大鼠的腹腔,连续测定腰椎骨密度、腰椎和股骨骨生物力学性能和骨代谢生化指标改变,光镜及电镜观察骨组织病理改变并进行形态计量学分析, ELISA法检测血清人降钙素浓度。探讨研究植入微囊化的人降钙素(hCT)cDNA转染细胞对去势大鼠的治疗效果,为临床应用基因工程细胞治疗绝经后骨质疏松提供新方法。
结果:⑴大鼠切除卵巢后12周,骨小梁数目减少,纤细,相互连接减弱,成功复制绝经后骨质疏松动物模型。去势大鼠体内降钙素基础值及其储备功能均低于对照组;体内甲状腺C细胞数目剧增。⑵阳性脂质体转染法将含人降钙素表达基因的质粒导入成肌细胞,G418加压筛选。RT-PCR检测从mRNA的水平证实染后的成肌细胞中有外源性人降钙素基因的转录;通过western blot和免疫组化的方法证实转染后的细胞有人降钙素蛋白表达的同时,ELISA法也可从五株抗性克隆培养上清中检测到一定水平的hCT,与对照组克隆相比差异有统计学意义。表明转染后的宿主细胞可以将胞质中合成的降钙素分泌至细胞外。⑶在体外、体内检测到人降钙素的持续分泌,说明重组人降钙素可渗透通过微囊壁,进而发挥其生理作用。⑷重组人降钙素显著促进体外培养成骨细胞的增殖、碱性磷酸酶的活性和矿化功能;呈剂量依赖关系。⑸重组人降钙素可减少破骨细胞数和骨吸收陷窝面积;呈剂量依赖性。⑹植入微囊化的人降钙素转染细胞后,去势大鼠骨转换率下降,改善骨小梁微结构,腰椎骨量和骨强度明显改善。对胫骨的松质骨量,部分地或有限度地防止骨丢失。
结论:⑴去势后大鼠甲状腺C细胞的分泌功能失代偿也许是骨量降低的原因之一。⑵微囊化hCT cDNA转染细胞能合成和向囊外分泌人降钙素。⑶重组人降钙素对体外培养成骨细胞的增殖、分化和矿化功能有明显促进作用;可减少破骨细胞的数量,抑制其骨吸收功能。⑷微囊化转基因修饰的成肌细胞分泌的重组人降钙素可防止去势后中轴骨的骨丢失。
Background: Osteoporosis is a disease of reduced skeletal mass and microarchitectural deterioration of the skeleton, resulting in an increased risk of fractures. Postmenopausal osteoporosis is the most common type of the disease. Fractures associated with osteoporosis and osteopenia are associated with significant increases in morbidity and mortality. Fractures resulting from low bone mass also have a significant impact on life quality of patients and social health care cost. The loss of ovarian hormone production in menopause is the major risk factor for osteoporosis, causing increased skeletal resorption and relatively decreased bone formation, continuing until the end of life. The increased bone remodeling, due to excessive osteoclast activity and an insufficiency of bone formation, results in deep resorption cavities, trabecular plate perforation, wide separation and disconnection of trabecular, and enlargement and coalescence of subendocortical spaces. Due to bone high-turnover rate after the menopause, antiresorptive drugs are reasonable choice for physicians. Several alternative treatments are available,including established drug therapies such as hormone replacement therapy,bisphosphonates, selective estrogen receptor modulators as well as calcitonin. Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals, which directly inhibits osteoclastic bone resorption. In spite of the novel therapeutic effect of calcitonin on bone diseases, the exact role of calcitonin in the pathogenesis of postmenopausal osteoporosis remains controversial. While , the use of these therapeutic proteins in the treatment of postmenopausal osteoporosis is limited by the need for repeated protein administration, costly production methods and antigenicity.
Objective: The purpose of the first part of this study was to investigate the changes of morphology and calcitonin secretory reserve of thyroid C cells in ovariectomized rats, in aiming to study the role of calcitonin in the pathogenesis of postmenopausal osteoporosis. Meanwhile,Mouse myoblasts strain which steadily expressed human calcitonin were established using the cationic liposome-based gene delivery technique. The hCT cDNA transfected myoblasts were encapsulated in non-antigenic biocompatible alginate-polylysine-alginate microcapsules. The effects of recombinant human calcitonin secreted from the cells on osteoblasts and osteoclasts were investigated in vitro. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats to examine the therapeutic feasibility of the protocol.
Method: Firstly, sixteen 6-month-old, female SD rats were divided into two groups. One was non-ovariectomized group (Sham) and the other was ovariectomized group (OVX). Twelve weeks after surgery, the alterations of thyroid C cell secretory reserve function in two groups were assessed by the calcium infusion test. A peroxidase–antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume、relative volume density and numerical density were evaluated in comparison with sham-operated control rats using a stereological method. Then, Mouse myoblasts were transfected with the cDNA for human calcitonin using the cationic liposome-based gene delivery technique and clones secreting high levels of human calcitonin were isolated. The expression and secretion of human calcitonin by myoblast cells was confirmed by RT-PCR、ELISA、Western blot analysis and immunohistochemical analyses. Upon enclosure in alginate-polylysine-alginate microcapsules, which are biocompatible membranes that permit exit of therapeutic protein but not entry of immune mediators, the encapsulated myoblasts were cultured in vitro. Primary osteoblasts and osteoclasts were subjected to recombinant human calcitonin secreted from the cells at different concentrations. The effects of recombinant peptide on proliferation and function of primary osteoblasts and osteoclasts were studied. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats. The concentration of human calcitonin was measured by ELISA. Bone mineral density of lumbar was determined by DEXA. Biomechanical property of lumbar and femur were also examined. Some biochemical markers of bone metabolism as serum calcium、serum inorganic phosphorus、serum osteocalcin (BGP) and unrinary pyridinoline (PYD) were examined. Bone histomorphometry、Light microscope and Scanning Electronic Microscopy were used to investigated skeletal changes in cancellous bone in section of lumbar and tibia.
Results:⑴Ovariectomy led to trabecular volume and number, wide separation and disconnection of trabecular in section of lumbar. The fact indicated the animal model of osteoporosis had been duplicated successfully. In ovariectomized rats, the three parameters as basal CT, the peak CT value and the CT increase range were significantly lower than those in normal controls. The number of thyroid C cells of the ovariectomized rats was markedly increased.⑵Reverse transcriptive-polymerase chain reaction (RT-PCR) and enzyme immunoassay confirmed that transgenes were expressed in the myoblast cells. Meanwhile, Western blot and immunohistochemical analyses detected significant increases in human calcitonin levels in the transfected myoblasts.⑶The encapsulated recombinant myoblasts continued to secrete human calcitonin in vivo and in vitro.⑷The recombinant protein showed marked increases in osteoblast proliferation, alkaline phosphates activity and formation of mineral nodules in dosage-dependant manner.⑸The recombinant human calcitonin decreased the number of osteoclasts and caused a dose-dependent decrease in mature osteoclasts activity as measured by the area of bone resorptive pits.⑹The bone turnover rate in ovariectomized rats had decreased after implantation. In comparison with the osteoporotic rats, those implanted with encapsulated recombinant myoblasts showed less bone loss; the reduction in bone loss was obvious in vertebral body, but this was hardly seen in the proximal tibial metaphases. Histological findings of lumbar vertebra revealed the improvement of bone architect of the axial bone.
Conclusion:⑴The deficiency of synthesis and release of calcitonin in the thyroid C cells may account partially for the bone loss in ovariectomized rats.⑵The encapsulated recombinant myoblasts can serve as an efficient and stable delivery vehicle producing human calcitonin.⑶The recombinant protein has positive effects on proliferation, ALP activity and mineralizing nodules formation in osteoblasts. It decreases the number of osteoclasts and inhibited their bone resorptive function.⑷The recombinant human calcitonin secreted from the encapsulated myoblasts can reverse the bone loss in axial bone due to ovariectomy.
目的:本研究首先拟观察去势大鼠基础值及其储备功能的改变;同时了解体内甲状腺C细胞形态变化特点。为进一步探讨降钙素在绝经后骨质疏松症发生中的作用提供试验依据。同时,拟微囊技术和转基因技术结合起来,通过脂质体转染方法建立降钙素基因高表达成肌细胞株,再将其微囊化,形成微囊化转降钙素基因修饰细胞。观察重组人降钙素对体外骨重建功能细胞的影响。进而将微囊化转基因修饰细胞移植体内,观察对去势大鼠的骨代谢与骨结构的影响。
方法:6月龄清洁级健康雌性SD大鼠16只,随机均分为去卵巢组和假手术组。去势12周后用钙负荷-降钙素兴奋试验分别观察两组降钙素基础值及其储备功能的改变。并处死每组大鼠,留取甲状腺组织,采用免疫组织化学方法显示降钙素阳性细胞并对其进行细胞形态计量学分析。另外,利用脂质体介导法将人降钙素基因(hCT)转染成肌细胞,以G418筛选出阳性克隆并扩大培养。用逆转录-聚合酶链反应(RT-PCR)、Western Blot和免疫组织化学分析目的基因及其蛋白表达;并用ELISA法检测上清中分泌的人降钙素浓度。将转染有hCT cDNA的L6成肌细胞包被在具有免疫隔离作用的海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊内形成微囊化基因转染细胞。在体外培养的同时,应用原代培养成骨细胞和破骨细胞方法,观察重组人降钙素对细胞形态、功能的影响。而后将微囊化基因转染细胞移植到大鼠的腹腔,连续测定腰椎骨密度、腰椎和股骨骨生物力学性能和骨代谢生化指标改变,光镜及电镜观察骨组织病理改变并进行形态计量学分析, ELISA法检测血清人降钙素浓度。探讨研究植入微囊化的人降钙素(hCT)cDNA转染细胞对去势大鼠的治疗效果,为临床应用基因工程细胞治疗绝经后骨质疏松提供新方法。
结果:⑴大鼠切除卵巢后12周,骨小梁数目减少,纤细,相互连接减弱,成功复制绝经后骨质疏松动物模型。去势大鼠体内降钙素基础值及其储备功能均低于对照组;体内甲状腺C细胞数目剧增。⑵阳性脂质体转染法将含人降钙素表达基因的质粒导入成肌细胞,G418加压筛选。RT-PCR检测从mRNA的水平证实染后的成肌细胞中有外源性人降钙素基因的转录;通过western blot和免疫组化的方法证实转染后的细胞有人降钙素蛋白表达的同时,ELISA法也可从五株抗性克隆培养上清中检测到一定水平的hCT,与对照组克隆相比差异有统计学意义。表明转染后的宿主细胞可以将胞质中合成的降钙素分泌至细胞外。⑶在体外、体内检测到人降钙素的持续分泌,说明重组人降钙素可渗透通过微囊壁,进而发挥其生理作用。⑷重组人降钙素显著促进体外培养成骨细胞的增殖、碱性磷酸酶的活性和矿化功能;呈剂量依赖关系。⑸重组人降钙素可减少破骨细胞数和骨吸收陷窝面积;呈剂量依赖性。⑹植入微囊化的人降钙素转染细胞后,去势大鼠骨转换率下降,改善骨小梁微结构,腰椎骨量和骨强度明显改善。对胫骨的松质骨量,部分地或有限度地防止骨丢失。
结论:⑴去势后大鼠甲状腺C细胞的分泌功能失代偿也许是骨量降低的原因之一。⑵微囊化hCT cDNA转染细胞能合成和向囊外分泌人降钙素。⑶重组人降钙素对体外培养成骨细胞的增殖、分化和矿化功能有明显促进作用;可减少破骨细胞的数量,抑制其骨吸收功能。⑷微囊化转基因修饰的成肌细胞分泌的重组人降钙素可防止去势后中轴骨的骨丢失。
Background: Osteoporosis is a disease of reduced skeletal mass and microarchitectural deterioration of the skeleton, resulting in an increased risk of fractures. Postmenopausal osteoporosis is the most common type of the disease. Fractures associated with osteoporosis and osteopenia are associated with significant increases in morbidity and mortality. Fractures resulting from low bone mass also have a significant impact on life quality of patients and social health care cost. The loss of ovarian hormone production in menopause is the major risk factor for osteoporosis, causing increased skeletal resorption and relatively decreased bone formation, continuing until the end of life. The increased bone remodeling, due to excessive osteoclast activity and an insufficiency of bone formation, results in deep resorption cavities, trabecular plate perforation, wide separation and disconnection of trabecular, and enlargement and coalescence of subendocortical spaces. Due to bone high-turnover rate after the menopause, antiresorptive drugs are reasonable choice for physicians. Several alternative treatments are available,including established drug therapies such as hormone replacement therapy,bisphosphonates, selective estrogen receptor modulators as well as calcitonin. Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals, which directly inhibits osteoclastic bone resorption. In spite of the novel therapeutic effect of calcitonin on bone diseases, the exact role of calcitonin in the pathogenesis of postmenopausal osteoporosis remains controversial. While , the use of these therapeutic proteins in the treatment of postmenopausal osteoporosis is limited by the need for repeated protein administration, costly production methods and antigenicity.
Objective: The purpose of the first part of this study was to investigate the changes of morphology and calcitonin secretory reserve of thyroid C cells in ovariectomized rats, in aiming to study the role of calcitonin in the pathogenesis of postmenopausal osteoporosis. Meanwhile,Mouse myoblasts strain which steadily expressed human calcitonin were established using the cationic liposome-based gene delivery technique. The hCT cDNA transfected myoblasts were encapsulated in non-antigenic biocompatible alginate-polylysine-alginate microcapsules. The effects of recombinant human calcitonin secreted from the cells on osteoblasts and osteoclasts were investigated in vitro. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats to examine the therapeutic feasibility of the protocol.
Method: Firstly, sixteen 6-month-old, female SD rats were divided into two groups. One was non-ovariectomized group (Sham) and the other was ovariectomized group (OVX). Twelve weeks after surgery, the alterations of thyroid C cell secretory reserve function in two groups were assessed by the calcium infusion test. A peroxidase–antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume、relative volume density and numerical density were evaluated in comparison with sham-operated control rats using a stereological method. Then, Mouse myoblasts were transfected with the cDNA for human calcitonin using the cationic liposome-based gene delivery technique and clones secreting high levels of human calcitonin were isolated. The expression and secretion of human calcitonin by myoblast cells was confirmed by RT-PCR、ELISA、Western blot analysis and immunohistochemical analyses. Upon enclosure in alginate-polylysine-alginate microcapsules, which are biocompatible membranes that permit exit of therapeutic protein but not entry of immune mediators, the encapsulated myoblasts were cultured in vitro. Primary osteoblasts and osteoclasts were subjected to recombinant human calcitonin secreted from the cells at different concentrations. The effects of recombinant peptide on proliferation and function of primary osteoblasts and osteoclasts were studied. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats. The concentration of human calcitonin was measured by ELISA. Bone mineral density of lumbar was determined by DEXA. Biomechanical property of lumbar and femur were also examined. Some biochemical markers of bone metabolism as serum calcium、serum inorganic phosphorus、serum osteocalcin (BGP) and unrinary pyridinoline (PYD) were examined. Bone histomorphometry、Light microscope and Scanning Electronic Microscopy were used to investigated skeletal changes in cancellous bone in section of lumbar and tibia.
Results:⑴Ovariectomy led to trabecular volume and number, wide separation and disconnection of trabecular in section of lumbar. The fact indicated the animal model of osteoporosis had been duplicated successfully. In ovariectomized rats, the three parameters as basal CT, the peak CT value and the CT increase range were significantly lower than those in normal controls. The number of thyroid C cells of the ovariectomized rats was markedly increased.⑵Reverse transcriptive-polymerase chain reaction (RT-PCR) and enzyme immunoassay confirmed that transgenes were expressed in the myoblast cells. Meanwhile, Western blot and immunohistochemical analyses detected significant increases in human calcitonin levels in the transfected myoblasts.⑶The encapsulated recombinant myoblasts continued to secrete human calcitonin in vivo and in vitro.⑷The recombinant protein showed marked increases in osteoblast proliferation, alkaline phosphates activity and formation of mineral nodules in dosage-dependant manner.⑸The recombinant human calcitonin decreased the number of osteoclasts and caused a dose-dependent decrease in mature osteoclasts activity as measured by the area of bone resorptive pits.⑹The bone turnover rate in ovariectomized rats had decreased after implantation. In comparison with the osteoporotic rats, those implanted with encapsulated recombinant myoblasts showed less bone loss; the reduction in bone loss was obvious in vertebral body, but this was hardly seen in the proximal tibial metaphases. Histological findings of lumbar vertebra revealed the improvement of bone architect of the axial bone.
Conclusion:⑴The deficiency of synthesis and release of calcitonin in the thyroid C cells may account partially for the bone loss in ovariectomized rats.⑵The encapsulated recombinant myoblasts can serve as an efficient and stable delivery vehicle producing human calcitonin.⑶The recombinant protein has positive effects on proliferation, ALP activity and mineralizing nodules formation in osteoblasts. It decreases the number of osteoclasts and inhibited their bone resorptive function.⑷The recombinant human calcitonin secreted from the encapsulated myoblasts can reverse the bone loss in axial bone due to ovariectomy.
引文
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