【作者】 胡洁；

【导师】 王润田； 裴雪涛；

【作者基本信息】 河北医科大学 ， 免疫学， 2009， 博士

【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(P<0.05)。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(P<0.05)、升高模型小鼠血清中肝功能指标ALT和AST含量(P<0.05)并促进肝组织的病理损伤(P<0.05)。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(P<0.05),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(P<0.05);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(P<0.05)、降低模型小鼠血清中肝功能指标ALT含量(P<0.05)并显著降低肝组织的病理损伤程度(P<0.05);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(P<0.05)并增加早期时相(3h)脾细胞Treg亚群细胞比例(P<0.05)。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(P<0.05);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(P<0.05);均可使MHCC/97H肿瘤细胞体外克隆形成能力显著降低(P<0.05);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(P>0.05)。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(P>0.05)。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。更多还原

【Abstract】 Obiectives: Have emerged as a promising therapeutic modality for tissue regeneration and repair, MSCs have low immunogenicity and the ability to migrate to sites of tissue injury and rapidly expand and differentiate along its own, allogeneil or xenogeneic lineages to repair the damaged tissue without rejection. Compare to the MSCs from adult, the human MSCs from fetus are present earlier during development, less likely to be infected with pathogen and can be more easily harvested. Therefore, MSCs from fetus are the main source for transplant.It is known that transplant between individuals with different genetic background can induce the rejection, which has not been solved perfectly until now. It hinders the clinical application of transplant. MSC transplant have generated expectations since MSCs are capable of differentiating along multiple lineages and have low immunogenicity.Numerous studies have demonstrated that MSCs have powerful immunosuppressive function, and may be involved in the mechanism of the vanishing of graft rejection. Therefore, it is very important to show that the engraft of ectogenic MSCs do not induce tumorigenesis.Up to now, MSCs are largely studied for their uses in tissue repair, immunotolerance reestablishment and other fields. The clinical trials of MSCs therapy in cardiac muscle repair, bone disease and metabolic disease have been carried out all over the world. Several studies indicated that adoption of autogeneic or allogenetic MSCs by different administration routes (region/i.v./i.p.) has therapeutic action on experimental autoimmune encephalomyelitis (EAE)and collagenⅡ-induced arthritis(CIA). Moreover, Le Blanc and his colleague have successfully harnessed MSCs to treat severe acute graft-versus-host disease(GVHD), and greatly reduced the incidence of GVHD. These results all suggest that MSCs could provide an ideal cell source in the treatment of immune-mediated diseases.In this study we examined the effect of hMSCs on ConA-induced hepatitis, and verified the therapeutic value and mechanism of hMSC in autoimmune hepatitis (AIH). Additionally, we validated the potential oncogenicity and promotion to hepatoma growth of hMSCs.Methods: hMSCs were isolated from aborted fetus’s bone marrow or amniotic fluid. In order to certifficate the cells cultured in vitro are hMSC, we observed the cell morphology under a phase microscope, detected the phenotype by flow cytometry (FCM) and identified the potential for differentiation by culture cells under conditions that favored adipogenic and osteogenic differentiation. To confirm the immunomodulatory effect of hMSCs isolated from aborted fetus’s bone marrow or amniotic fluid in vitro using MTS. Splenocytes stimulated by ConA were incubated in medium, the medium supernatant was harvested. hMSCs were incubated in the supernatant, and its features including proliferation, phenotype and chromosome caryotype were observed. ConA mice model were performed according to the protocol of Tiegs. The indexes of hepatic injury included the serum levels of alanine aminotransferase (ALT) , aspartate aminotransferase (AST) and C3, and the pathological lesion of liver. We used the indexes above to verify the effect of hMSCs with or without pretreated to the ConA mice model. To test if mice splenocytes cultivate supernatant pretreatment could induce the malignant growth of hMSCs, hMSCs with pretreated were subcutaneously inoculated in athymic mice. To test the influence of hMSCs to tumor, we used MHCC/97H (human hepatoma carcinoma cell) and H22(mouse hepatoma carcinoma cell) as the target cells of hMSC with or withou pretreated and detected the cell cycle, proliferation and the capability of invasion in vivo or in vitro.Results:1 The preparation and identification of hMSCs and its Immunosuppression. hMSCs were isolated from aborted fetus’s bone marrow or amniotic fluid, and then was cultured as adherent cells. The cell of passage 3-5 displayed a morphology and phenotype typical of MSC, showing the potential for differentiation into adipogenic, chondrogenic and osteogenic cells.Compared to the control group, the proliferation of splenocytes induced by ConA in the hMSC-treated group was significantly suppressed in a dose-dependent fashion(P<0.05).2 hMSCs without pretreated can exacerbate the immunologic liver injury in ConA micehMSCs without pretreated could cut down the survival rate (P<0.05), heighten the serum content of ALT and AST (P<0.05) and intensify the ConA-induced liver injury (P<0.05).These findings indicated hMSCs without pretreated could exacerbate the immunologic liver injury in ConA mice.3 The promotion to immunologic injury induced by ConA of hMSCs without pretreated were independent on the expression of HLA-A,B,C or the serum content of C3HLA-A,B,C(-)/HLA-DR(-) and HLA-A,B,C(+)/HLA-DR(-) hMSCs without pretreated could both significantly increase the serum ALT levels(P<0.05). These results indicated the promotion to immunologic injury of hMSCs without pretreated were independent on the expression of HLA-A,B,C.Multiple linear regression was used to analyze the relationship between C3 and ALT, excluding the influence of time and group, there was no relationship between ALT and C3 (P>0.05). The above result showed that the promotion to immunologic injury of hMSCs without pretreated was independent on the serum contents of C3.4 hMSCs without pretreated can induce light damage of liver cells in normal miceCompared with the NS/NS group,the focus lytic necrosis, apoptotic body and inflammation in the district of portal in the ambitus of acini hepatis and the district of portal in the NS/na?ve hMSC group were different(P<0.05), indicated that hMSCs without pretreated could induce light damage of liver cells in normal mice.5 hMSCs with pretreated can extenuate the immunologic liver injury in ConA micehMSCs with pretreated could increase the survival rate (P<0.05), cut down the serum content of ALT and AST (P<0.05) and extenuate the ConA-induced liver injury (P<0.05). These findings indicated hMSCs with pretreated could protect the liver injury ConA mice .6 The decrease of FasL expression on hepatic tissue and the increase in the Treg cell ratio in splenocytes may involve in the protective effect of hMSCs with pretreated.hMSCs with pretreated can suppress the expression of FasL (P<0.05) and increase the ratio of Treg cell in splenocytes in the earlier period(3h) (P<0.05).7 The expression of IL-6, IL-8 and RANTES were increased in hMSCs with pretreated.By detecting the expression of inflammatory factor in the cultivate supernatant of hMSCs with or without pretreated by antibody array, we could find that the hMSC with pretreated expressed more IL-6, IL-8 and RANTES than hMSC without pretreated, and it may involve the protective effect of hMSCs with pretreated.8 Pretreatment could not induce the malignant growth of hMSCsThe hMSCs with pretreated by mice splenocytes cultivate supernatant has the same phenotype(CD29+, CD44+, CD90+, CD105+;HLA-DR-, CD14-, CD19-, CD34-, CD45-, CD31-) and caryotype of chromosome (2n=46)with hMSCs with or without pretreated proliferate arrest by blocking cell cycle at G0/G1 stage(P<0.05). Subcutaneous inoculation of hMSCs with pretreated in athymic mice, and no tumor emerge. So pretreated hMSCs by mice splenocytes cultivate supernatant could not induce the malignant growth of hMSC. 9 The effect of hMSCs with or without pretreated on the growth of hepatica cells in vivo or vitrohMSCs with or without pretreated can induce the MHCC/97H or H22 cells by blocking cell cycle at G0/G1 stage (P<0.05), and the inhibitory intensity between two type hMSC was not statistically significant (P>0.05). And hMSCs with or without pretreated had nothing to do with the capability of migration in vitro of MHCC/97H cell (P>0.05).Subcutaneous inoculation of MHCC/97H and H22 in athymic and BABL/c mice respectively, then hMSCs with or without pretreated. hMSCs with or without pretreated had nothing to do with the life span, tumor growth and capability of migration of tumor cell in vivo(P>0.05).Conclusions:1 Use of adherence-digest-serial subcultivation can harvest functional hMSCs successfully.2 hMSCs inhibit the proliferation of T cells stimulated by ConA.3 As we know, this is the first report that hMSCs with or without pretreated has opposite function on the ConA mice.4 hMSCs without pretreated can significantly exacerbate the immunologic liver injury in ConA mice.5 hMSCs with pretreated can powerfully protect the ConA mice.6 The promotion to immunologic injury of ConA mice by hMSCs without pretreated is independent on the expression of HLA-A,B,C and the serum content of C3.7 The decrease of FasL expression on hepatic tissue and the increase in the Treg cell ratio in splenocytes are related to the protective effect of hMSCs with pretreated.8 The expression of IL-6, IL-8 and RANTES is increased in hMSC with pretreated.9 hMSCs isolated from aborted fetus’s bone marrow does not have the risk of tumorigenesis.更多还原