人胎儿源间充质干细胞的分离鉴定及其在免疫性肝损伤中的免疫双效作用研究

发布时间：2018-06-08 03:41

  本文选题：间充质干细胞 + 免疫性肝损伤　； 参考：《河北医科大学》2009年博士论文

【摘要】： 目的:近年来,间充质干细胞(mesenchymal stem cell,MSC)以其能在自体、同种异体甚至异种异体体内,旺盛地增殖和多向地分化,有效修复甚至完全重建功能严重受损甚或几近缺如的组织器官而不被排斥,已经成为组织修复和再生重建领域内备受关注的研究热点。在人源MSC中,胎儿源MSC与成人源者相比,因其发育上更早、更少被病原体感染、安全性更高、获取和制备相对更为捷易等优点,更是成为人们多愿选择的来源。 现代免疫学研究已经揭示,自体和同基因个体(如同卵孪生和纯系内个体)间的组织器官移植之所以不被排斥,是因供体与受体的组织相容性抗原一致或几近一致所致。而同种异体间,尤其是异种异体间的组织器官移植一定会被排斥,是因供体和受体间组织相容性抗原不一致而致,且两者间的组织相容性抗原相差越远,则排斥反应的发生就越迅速、越强烈。由是,在人类为修复或重建功能已严重受损甚至已几近缺如的组织器官,除同卵孪生外,因实难觅组织相容性抗原一致或几近一致的供体,不得已而在移植后必须长期应用昂贵的免疫抑制剂维持,但随之又会带来易发严重感染甚至肿瘤的危险。由此,既不难理解为何人们会对人源尤其是胎儿源MSC寄予厚望而备受关注,同时也强烈提示,同种异体特别是异种异体MSC移植不被排斥,是否是其本身具有介导和启动有效免疫抑制作用机制所致;如是,则在其应用之后是否有抑制机体免疫功能特别是招致肿瘤发生的风险性,势必就成为该领域中必须予以高度重视和深入探索的重要课题。 迄今,已有许多研究小组对MSC在组织修复和免疫耐受重建等领域的实验研究和临床应用,进行了多方面的大胆探索和勇敢尝试。有关心肌修复、骨疾病及代谢性疾病的MSC细胞治疗的临床试验已在世界各地先后开展;而Le Blanc及其同事应用同种异体MSC于移植物抗宿主病(graft versus host disease, GVHD)的大胆尝试,更坚定了人们将其应用于免疫损伤性疾病治疗的信心。还有将自体或同种异体MSC以不同给药方式(局部、静脉和腹腔)治疗实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)及Ⅱ型胶原诱导的类风湿性关节炎(collagenⅡ-induced arthritis, CIA)模型的研究均获成功。更兼我们惊奇地发现经与未经预处理的人胎儿源MSC,对ConA诱导的免疫性肝损伤有双效作用,因这确是在世界上尚无报道,而极大地激发了我们对人胎儿源MSC免疫生物学特性及其与肿瘤免疫学关系的浓厚研究兴趣。 为此,本课题分离鉴定了胎儿骨髓源和羊水源MSC以优化来源,研究其免疫抑制功能特别是对ConA诱导免疫性肝损伤的双效作用,在分析其介导的免疫抑制分子的基础上,探索其之致瘤的风险性,为MSC的进一步研究和应用提供新的理论和实验依据。 方法:贴壁消化传代培养法,自人胎儿骨髓及羊水细胞获取,并经形态学、细胞表面标记和分化潜能鉴定,确系为人胎儿源MSC(以下简写为hMSC)。MTS法评价hMSC对ConA诱导小鼠脾细胞增殖的抑制作用。获取ConA刺激小鼠脾细胞增殖的培养上清,以其培养hMSC而获取经预处理的hMSC;观察其生长增殖、细胞表型、染色体核型等细胞生物学特性。参照Tiegs等的方法建立ConA诱导免疫性肝损伤动物模型,肝损伤指标包括小鼠存活率、血清谷丙转氨酶(glutamate-pyruvate transaminase,ALT)和谷草转氨酶(glutamic-oxalacetic transaminase,AST)含量以及肝组织病理损伤情况等。以模型动物肝损伤指标为对照,观察经或未经预处理的hMSC对模型动物肝损伤的影响,评价其对肝损伤的保护或促进作用。裸鼠皮下移植,排除hMSC成瘤可能;选择人肝癌细胞系MHCC/97H肿瘤细胞和小鼠肝癌细胞系H22肿瘤细胞为对象,观察hMSC对肿瘤细胞体内/外细胞周期、体外克隆形成能力、荷瘤小鼠生存期、皮下移植瘤体内生长增殖以及体内/外侵袭转移等的影响,评价其致瘤的风险性。 结果: 1人胎儿骨髓源及羊水源MSC的分离鉴定及其免疫抑制作用 贴壁消化传代培养法,自人胎儿骨髓及羊水细胞获取,连续传代培养3~5代后,其形态学、细胞表面标记和分化潜能鉴定,均具人MSC典型特征,确系获得了合格的hMSC。 ConA刺激小鼠脾细胞增殖实验显示,hMSC有显著的、剂量依赖式的免疫抑制作用(P0.05)。 2未预处理的hMSC对ConA诱导小鼠免疫性肝损伤的作用 未预处理的hMSC可显著降低ConA诱导免疫性肝损伤模型小鼠存活率(P0.05)、升高模型小鼠血清中肝功能指标ALT和AST含量(P0.05)并促进肝组织的病理损伤(P0.05)。显示未预处理的hMSC对ConA诱导免疫性肝损伤有显著的促进作用。 3未预处理的hMSC对模型小鼠肝损伤的促进作用与HLA分子表达及补体C3介导的关系 HLA-A,B,C(+)/HLA-DR(-)及HLA-A,B,C(-)/HLA-DR(-)的hMSC均可显著升高模型小鼠血清中肝损伤指标ALT的含量(P0.05),显示未预处理的hMSC对模型小鼠肝损伤的促进作用,与其表面HLA分子表达无关;各组小鼠血清ALT和补体C3含量测定及回归分析,显示未预处理的hMSC促进模型小鼠肝损伤亦与补体C3介导无关。 4未预处理的hMSC对正常小鼠肝脏组织的影响 小鼠肝组织病理切片HE染色结果显示,未预处理的hMSC组与正常组小鼠相比,肝小叶周边、界板肝细胞及汇管区的点灶状溶解性坏死、凋亡小体及汇管区炎等病理指标差异明显(P0.05);提示未预处理的hMSC腹腔注射可致正常小鼠肝组织轻微病理损伤。 5经预处理的hMSC对ConA诱导小鼠免疫性肝损伤的作用。 经预处理的hMSC可显著升高ConA诱导免疫性肝损伤模型小鼠存活率(P0.05)、降低模型小鼠血清中肝功能指标ALT含量(P0.05)并显著降低肝组织的病理损伤程度(P0.05);显示经预处理的hMSC对ConA诱导免疫性肝损伤有显著的保护作用。 6经预处理的hMSC对模型小鼠肝组织FasL表达和脾Treg亚群细胞比例的影响。 经预处理的hMSC可显著减少模型组小鼠肝组织FasL表达(P0.05)并增加早期时相(3h)脾细胞Treg亚群细胞比例(P0.05)。提示使模型鼠肝组织FasL表达减少和脾Treg细胞比例增加,可能是经预处理的hMSC对模型小鼠免疫性肝损伤具有保护作用的分子机制。 7经与未经预处理的hMSC相关细胞因子分泌差异 经预处理的hMSC其IL-6、IL-8及RANTES等细胞因子分泌,较未预处理的hMSC增多。提示,经预处理的hMSC有可能藉增加这些细胞因子的分泌,参与对ConA诱导免疫性肝损伤的保护作用。 8经预处理的hMSC的自身成瘤性 以ConA刺激小鼠脾细胞增殖的培养上清对hMSC行预处理后, hMSC的细胞周期被阻于G0/G1期,增殖指数显著降低(P0.05);细胞表型和染色体核型保持不变;接种于裸鼠皮下,亦无肿瘤形成。提示预处理未导致hMSC的自身成瘤性。 9经预处理的hMSC的促瘤性 经与未经预处理的hMSC,均可使人肝癌细胞系MHCC/97H肿瘤细胞及小鼠肝癌细胞系H22肿瘤细胞阻滞于G0/G1期,显著降低两种肿瘤细胞的增殖指数(P0.05);均可使MHCC/97H肿瘤细胞体外克隆形成能力显著降低(P0.05);预处理与否,不影响hMSC对这两种肿瘤细胞发挥上述这些抑制作用的强度(P0.05)。经与未经预处理的hMSC,对MHCC/97H肿瘤细胞和H22肿瘤细胞体内移植瘤的生长增殖、体内/外侵袭转移及荷瘤小鼠生存期无影响(P0.05)。提示预处理未导致hMSC的促瘤性。 结论: 1贴壁消化传代培养法可有效从骨髓和羊水,获取合格的人胎儿源MSC。 2人胎儿源MSC对ConA刺激的T细胞增殖有显著的免疫抑制作用。 3经或未经ConA刺激T细胞增殖的培养上清预处理,可显著改变人胎儿源MSC对ConA诱导免疫性肝损伤的影响模式,此结果与结论国内外未见报道。 4未预处理的人胎儿源MSC对ConA诱导免疫性肝损伤有显著的促进作用。 5经预处理的人胎儿源MSC对ConA诱导免疫性肝损伤有显著的保护作用。 6未预处理的人胎儿源MSC对ConA诱导免疫肝损伤的促进作用与其表面HLA分子表达及补体C3的介导作用无关。 7经预处理的人胎儿源MSC对ConA诱导免疫肝损伤的保护作用与其致肝组织FasL表达降低及脾细胞早期时相(3h)Treg亚群细胞比例增高有关。 8经预处理的人胎儿源MSC,其IL-6、IL-8及RANTES细胞因子表达增加。 9人胎儿源MSC在观察期内未见成瘤性和促瘤活性,初步判断其在所观察期内不具有致瘤风险性。
[Abstract]:Objective: in recent years, mesenchymal stem cell (MSC) has become a field of tissue repair and regenerative reconstruction with its ability to proliferate and multidirectional differentiation in autologous, allogeneic and heterogeneous allogeneic bodies, to effectively repair and even completely reconstruct the tissues and organs that have been severely damaged or almost absent. In human source MSC, fetal source MSC, compared with adult source, has the advantages of earlier development, less infection by pathogens, higher safety, and relatively easy access to preparation and preparation, and is also the source of people's choice.
Modern immunological studies have revealed that tissue and organ transplantation between autologous and homologous individuals (like egg twins and individuals in pure lines) is not excluded because of the consistent or close agreement of the donor and receptor histocompatibility antigen, and the allograft, especially the allogeneic allograft, will be rejected. Due to the inconsistency of the histocompatibility antigen between the donor and the recipient, and the farther the difference of the histocompatibility antigen between the two is, the more rapid and stronger the rejection occurs. A consistent or nearly consistent donor must not be used for long-term application of expensive immunosuppressive agents, but it will also bring about the risk of severe infection or even cancer. Thus, it is not difficult to understand why people are highly concerned about the expectations of human sources, especially fetal MSC, and also strongly suggest that the allograft is special. Not heterologous allograft MSC transplantation is not rejected, whether it has the mechanism to mediate and activate the effective immunosuppressive mechanism. If, if it has the risk of inhibiting the body's immune function, especially the occurrence of tumor, it is bound to be an important subject in this field.
So far, many research teams have made bold and bold attempts at the experimental research and clinical application of MSC in the fields of tissue repair and immune tolerance reconstruction. The clinical trials of MSC cell therapy for myocardial repair, bone disease and metabolic diseases have been carried out all over the world; and Le Blanc and its colleagues should A bold attempt to use allogeneic MSC for graft versus host disease (GVHD), and to strengthen its confidence in the treatment of immune injury diseases, and the treatment of experimental autoimmune encephalomyelitis (experimental auto) by the use of autologous or allogenic MSC in different ways (local, vein and abdominal cavity) Immune encephalomyelitis, EAE) and type II collagen induced rheumatoid arthritis (collagen II -induced arthritis, CIA) have been successfully studied. Moreover, we were surprised to find that the untreated human fetal MSC, which has double effects on the immune liver injury induced by ConA, is not yet reported in the world. The earth has stimulated our interest in studying the immunological characteristics of human fetal MSC and its relationship with tumor immunology.
Therefore, we isolated and identified the fetal bone marrow source and sheep water source MSC to optimize the source and study the double effect of its immunosuppressive function, especially on ConA induced immune liver injury. On the basis of the analysis of its mediated immunosuppressive molecules, the risk of its tumor induced tumor was explored, and a new theory for further research and application of MSC was provided. Experimental basis.
Methods: human fetal bone marrow and amniotic fluid cells were obtained from human fetal bone marrow and amniotic fluid cells, and were identified by morphology, cell surface labeling and differentiation potential. The inhibitory effect of hMSC on the proliferation of spleen cells induced by ConA was evaluated by human fetal MSC (below hMSC).MTS method. The culture supernatant of spleen cell proliferation stimulated by ConA was obtained. HMSC was cultured and pretreated hMSC was obtained, and the cell biological characteristics of its growth, proliferation, cell phenotype, chromosome karyotype and so on were observed. The animal models of immune liver injury induced by ConA were established with reference to Tiegs, including the survival rate of the mice, the serum alanine transaminase (ALT), and the transglutaminate of cereal grass. Enzyme (glutamic-oxalacetic transaminase, AST) content as well as pathological damage of liver tissue. The effect of hMSC on liver injury of model animals was observed or untreated by the model animal liver damage index, and the protective or promoting effect on liver injury was evaluated. The subcutaneous transplantation of nude mice, the possibility of removing hMSC into the tumor, was used to select human liver cancer. The cell line MHCC/97H tumor cells and the mouse liver cancer cell line H22 tumor cells were used to observe the effects of hMSC on the cell / outer cell cycle of the tumor cells, the ability of in vitro cloning and formation, the survival period of the tumor bearing mice, the growth and proliferation of the subcutaneous transplanted tumor and the invasion and metastasis of the body / outside, and to evaluate the risk of the tumor.
Result:
Isolation, identification and immunosuppressive effect of MSC from 1 human fetal bone marrow and sheep water sources
After being obtained from human fetal bone marrow and amniotic fluid cells from human fetal bone marrow and amniotic fluid cells, after continuous subculture of 3~5 generation, the morphology, cell surface labeling and differentiation potential were identified. All of them have the typical characteristics of human MSC, and the qualified hMSC. is obtained.
ConA stimulated splenocytes proliferation in mice showed that hMSC had a significant dose-dependent immunosuppressive effect (P0.05).
2 the effect of untreated hMSC on ConA induced immune liver injury in mice
Untreated hMSC could significantly reduce the survival rate of ConA induced immune liver injury model mice (P0.05), increase the liver function index ALT and AST content (P0.05) in the serum of the model mice and promote the pathological damage of liver tissue (P0.05). It showed that the untreated hMSC had a significant effect on the immune liver injury induced by ConA.
3 the effect of untreated hMSC on liver injury in mice and the expression of HLA and complement C3.
HLA-A, B, C (+) /HLA-DR (-) and HLA-A, B, C (-) /HLA-DR (-) hMSC can significantly increase the content of the liver damage index ALT content (P0.05) in the serum of the model mice, showing that the unpretreated hMSC is not related to the expression of the liver damage in the model mice. The treatment of hMSC promoted liver damage in model mice was also independent of complement C3 mediating.
4 Effect of untreated hMSC on liver tissue in normal mice
The results of HE staining in the pathological section of liver tissue in mice showed that compared with the normal group, the hMSC group of the untreated group was significantly different from the normal group of the liver lobules, the focal dissolving necrosis of the focal liver cells and the sink area, the apoptotic bodies and the manifold inflammation (P0.05), suggesting that the untreated hMSC could lead to a slight liver tissue in normal mice. Pathological injury.
5 the effect of pretreated hMSC on ConA induced immune liver injury in mice.
The pretreated hMSC significantly increased the survival rate of ConA induced immune liver injury model mice (P0.05), reduced the liver function index ALT content (P0.05) in the serum of the model mice and significantly reduced the degree of pathological damage of liver tissue (P0.05), and showed that the pretreated hMSC had a significant protective effect on ConA induced immune liver injury.
6 the effect of pretreated hMSC on the expression of FasL and the proportion of Treg subsets in liver tissue of model mice.
The pretreated hMSC could significantly reduce the FasL expression of liver tissue in the model mice (P0.05) and increase the Treg subgroup ratio (P0.05) in the early phase (3H) of the spleen cells (P0.05), suggesting that the reduced expression of FasL in the liver tissue of the model mice and the proportion of the spleen Treg cells increased, which may be a protective molecule of the pretreated hMSC on the immune liver damage in the model mice. Mechanism.
7 differences in hMSC related cytokine secretion between pretreated and untreated
The pretreated hMSC, such as IL-6, IL-8 and RANTES, is more secreted than untreated hMSC. It is suggested that the pretreated hMSC may be involved in the protection of ConA induced immune liver injury by increasing the secretion of these cytokines.
8 pretreated hMSC's self tumorigenicity
The cell cycle of hMSC was pretreated with ConA stimulated the proliferation of mouse splenocytes, the cell cycle of hMSC was blocked at G0/G1, the proliferation index decreased significantly (P0.05), the cell phenotype and chromosome karyotype remained unchanged, and no tumor was formed in subcutaneous inoculation of nude mice, suggesting that preconditioning did not lead to the self tumorigenicity of hMSC.
9 pretreated hMSC's tumorigenicity
The hMSC tumor cells of human hepatoma cell line MHCC/97H and H22 tumor cell line H22 were blocked at G0/G1 stage, and the proliferation index of two tumor cells was significantly reduced (P0.05), and the ability of MHCC/97H tumor cells to be cloned in vitro was significantly reduced (P0.05). The preconditioning did not affect hMSC against this two. The growth and proliferation of tumor cells in MHCC/97H tumor cells and H22 tumor cells, in vivo / out invasion and metastasis and the survival period of tumor bearing mice (P0.05) were not affected by pretreated hMSC (P0.05). It suggested that the preconditioning did not lead to the tumor growth of hMSC in the tumor cells (P0.05).
Conclusion:
1 adherent digestion and subculture can effectively obtain MSC. from human bone marrow and amniotic fluid.
The 2 fetal fetal MSC has significant immunosuppressive effect on the proliferation of T cells stimulated by ConA.
3 the preconditioning of cultured supernatant with or without ConA stimulation of T cell proliferation can significantly change the model of human fetal source MSC on ConA induced immune liver injury. The results and conclusions have not been reported at home and abroad.
4 untreated human fetal MSC has a significant effect on ConA induced immune liver injury.
5 pretreated human fetal MSC has a significant protective effect on ConA induced immune liver injury.
6 the effect of unpretreated human fetal MSC on ConA induced liver injury is not related to the expression of HLA on the surface and the mediating effect of complement C3.
7 the protective effect of pretreated human fetal MSC on ConA induced immune liver injury is related to the decrease of FasL expression in liver tissue and the increase in the proportion of Treg subgroup cells in the early phase of the spleen cells (3H).
8 the expression of IL-6, IL-8 and RANTES cytokines increased in pretreated human fetal MSC.
There was no tumorigenic and tumorigenic activity in the 9 fetal fetal MSC during the observation period.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R575;R329

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