血管外膜平滑肌祖细胞在移植物血管病变中的作用及机制研究

发布时间：2018-06-12 17:17

本文选题：移植物血管病变 + 内膜增生　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分移植物血管外膜平滑肌祖细胞的分离、鉴定及诱导分化目的：为了确定移植物血管病变内膜平滑肌样细胞来源,并了解其生物学特性。方法：将C57BL/6背景的eGFP转基因鼠与野生型C57BL/6进行交叉骨髓移植后作为主动脉移植的受体,然后将BALB/C供体小鼠的胸主动脉移植入受体腹主动脉之间。移植术后于1、2、4、6、8、10周等不同时间点取出移植血管进行免疫荧光检测。使用流式分选技术将移植物血管中内膜或外膜的eGFP阳性细胞分选出来在体外培养,并诱导分化,通过免疫荧光技术鉴定其分子标志等。将体外培养纯化的带有eGFP荧光标记的平滑肌祖细胞包裹在普通野生型C57BL/6移植血管周围,了解其能否迁移入血管内膜。结果：骨髓来源的单核细胞在术后两周在内膜中达到高峰,随后逐渐下降,但并不能表达平滑肌标志物SM-MHC,可表达CD68等单核/巨噬细胞标志。而在术后两周移植血管外膜中开始出现一群非骨髓来源的细胞,可表达干细胞标志Sca-1及早期平滑肌分化标志SM-22α等,但不能表达SM-MHC。该群细胞可迁移进入血管内膜逐渐表达SM-MHC,并成为增生内膜的主要细胞。通过流式分选技术从血管外膜将该群细胞分选后并在体外培养发现在体外经过PDGF-BB或TGF-β诱导可表达SM-MHC。再体外培养纯化的平滑肌祖细胞可从外膜迁移入内膜并加重移植物血管病变的内膜增生。结论：移植后受体的中非骨髓细胞来源的平滑肌祖细胞可迁移入血管内膜并分化为表达SM-MHC的平滑肌样细胞,从而促进内膜增生病变。第二部分miR-155可调控单核细胞趋化蛋白-1(MCP-1)浓度梯度诱导外膜血管平滑肌祖细胞向内膜定向迁移目的：了解miR-155在移植物血管病变中的作用以及对骨髓来源的单核细胞和平滑肌祖细胞的功能影响。方法：将C57BL/6背景的miR-155基因敲除鼠和野生型C57BL/6小鼠进行交叉骨髓移植后再作为主动脉移植的受体。流式细胞仪分选出移植物血管中骨髓来源的单核细胞,使用RT-PCR方法测定各种趋化因子的变化水平,并转染miR-155mimics和inhibitor后使用ELISA测定培养上清细胞因子变化情况。使用RT-PCR方法测定移植血管标本内膜外膜间趋化因子浓度梯度水平,并通过Transwell实验测定在骨髓来源的单核细胞对平滑肌祖细胞迁移能力的诱导作用及机制。结果：术后八周我们发现接受了miR-155敲除鼠骨髓细胞的小鼠内膜增生病变最轻,而即使是miR-155敲除鼠接受了miR-155+/+骨髓细胞小鼠也发生明显内膜增生。miR-155可通过抑制骨髓来源单核细胞B细胞淋巴瘤因子6的表达而促进单核细胞趋化因子-1(MCP-1)的表达从而诱导平滑肌祖细胞的发生定向迁移。当骨髓来源的单核细胞中miR-155被敲除后在移植血管内膜外膜间MCP-1浓度梯度大幅度降低以至于难以形成有效的浓度梯度。结论：miR-155可通过调控骨髓来源单核细胞功能分泌MCP-1而改变内膜外膜之间浓度梯度,促进平滑肌祖细胞向内膜迁移。第三部分TGF-βI型受体磷酸激酶抑制剂(SD-208)可抑制平滑肌祖细胞的增殖和迁移能力目的：评价TGF-βI型受体磷酸化酶抑制剂(SD-208)能否抑制内膜平滑肌样细胞的增殖及迁移能力而减轻移植物血管病变。方法：BALB/c小鼠主动脉移植到C57BL/6小鼠腹主动脉,术后每天给予40或60mg/kg的SD-208灌胃,术后1、2、4、6、8周取出移植血管分析内膜增生程度并通过免疫组化方法分析TGF-β/Smad3通路各蛋白表达情况。体外培养中层平滑肌细胞及内膜平滑肌样细胞使用TGF-β and SD-208对于两种细胞进行干预,了解增殖、迁移能力的异同。结果：在给予40及60mg/kg等不同剂量的SD-208治疗后,移植血管的内膜增生程度分别比对照组下降了32%和48%。SD-208可以抑制TGF-p对于内膜平滑肌样细胞增殖和迁移能力的促进作用而对于其对于中层平滑肌细胞的抑制作用并没有明显影响。SD-208可明显减低结缔组织生长因子的表达水平。在内膜平滑肌样细胞中Smad3的水平明显高于中层平滑肌细胞结论：SD-208可有效抑制内膜平滑肌样细胞的增殖和迁移能力,减少细胞外基质的分泌。
[Abstract]:Isolation , identification and induction of vascular smooth muscle progenitor cells from the first part of the graft

Objective : To determine the origin of vascular smooth muscle - like cells and to understand its biological characteristics .

Methods : The eGFP transgenic mice of C57BL / 6 background and wild type C57BL / 6 mice were cross - linked with wild - type C57BL / 6 as the recipient of aortic graft , and then transplanted into the abdominal aorta of recipient abdominal aorta at different time points , such as 1 , 2 , 4 , 6 , 8 , 10 weeks after transplantation .

Results : The bone marrow - derived mononuclear cells reached the peak at two weeks after operation and then gradually decreased , but it was not possible to express SM - MHC of smooth muscle marker , which could express SM - MHC .

CONCLUSION : Smooth muscle progenitor cells derived from non - bone marrow cells from post - transplant recipients can migrate into the intima and differentiate into smooth muscle - like cells expressing SM - MHC so as to promote intimal hyperplasia .

The second part miR - 155 regulates monocyte chemoattractant protein - 1 ( MCP - 1 ) concentration gradient to induce the migration of exogenous membrane vascular smooth muscle progenitor cells to the intima .

Objective : To investigate the role of miR - 155 in vascular diseases of graft and the functional effects of miR - 155 on bone marrow - derived monocytes and smooth muscle progenitor cells .

Methods : C57BL / 6 mouse and wild - type C57BL / 6 mice were crossed with bone marrow from C57BL / 6 mice .

Results : The expression of miR - 155 knockout mice in mouse bone marrow cells was the lightest in eight weeks after operation , and even if miR - 155 knockout mice received miR - 155 + / + bone marrow cells . miR - 155 can promote the expression of monocyte chemoattractant protein - 1 ( MCP - 1 ) to induce targeted migration of smooth muscle progenitor cells .

Conclusion : miR - 155 can change the concentration gradient between the inner membrane and the outer membrane by regulating the function of MCP - 1 secreted by the monocyte in the bone marrow , and promote the migration of smooth muscle progenitor cells to the intima .

The third part TGF - 尾I receptor phosphokinase inhibitor ( SD - 208 ) can inhibit the proliferation and migration of smooth muscle progenitor cells .

Objective : To evaluate whether TGF - 尾I receptor phosphorylase inhibitor ( SD - 208 ) can inhibit the proliferation and migration of smooth muscle - like cells and reduce graft vascular disease .

Methods : BALB / c mouse aorta was transplanted to the abdominal aorta of C57BL / 6 mice . SD - 208 was given 40 or 60 mg / kg daily after operation . The expression of TGF - 尾 / Smad3 was analyzed by immunohistochemistry . The expression of TGF - 尾 / Smad3 was analyzed by immunohistochemistry . TGF - 尾 and SD - 208 were used to intervene the two kinds of cells in vitro .

Results : After treatment with different doses of SD - 208 at doses of 40 and 60 mg / kg , the degree of intimal hyperplasia of transplanted blood vessels decreased by 32 % and 48 % respectively than in the control group .

Conclusion : SD - 208 can effectively inhibit the proliferation and migration of smooth muscle - like cells and reduce the secretion of extracellular matrix .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R654.3

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