本文選題：移植物血管病變 + 內(nèi)膜增生��； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分移植物血管外膜平滑肌祖細(xì)胞的分離、鑒定及誘導(dǎo)分化 目的：為了確定移植物血管病變內(nèi)膜平滑肌樣細(xì)胞來源,并了解其生物學(xué)特性。 方法：將C57BL/6背景的eGFP轉(zhuǎn)基因鼠與野生型C57BL/6進(jìn)行交叉骨髓移植后作為主動脈移植的受體,然后將BALB/C供體小鼠的胸主動脈移植入受體腹主動脈之間。移植術(shù)后于1、2、4、6、8、10周等不同時間點取出移植血管進(jìn)行免疫熒光檢測。使用流式分選技術(shù)將移植物血管中內(nèi)膜或外膜的eGFP陽性細(xì)胞分選出來在體外培養(yǎng),并誘導(dǎo)分化,通過免疫熒光技術(shù)鑒定其分子標(biāo)志等。將體外培養(yǎng)純化的帶有eGFP熒光標(biāo)記的平滑肌祖細(xì)胞包裹在普通野生型C57BL/6移植血管周圍,了解其能否遷移入血管內(nèi)膜。 結(jié)果：骨髓來源的單核細(xì)胞在術(shù)后兩周在內(nèi)膜中達(dá)到高峰,隨后逐漸下降,但并不能表達(dá)平滑肌標(biāo)志物SM-MHC,可表達(dá)CD68等單核/巨噬細(xì)胞標(biāo)志。而在術(shù)后兩周移植血管外膜中開始出現(xiàn)一群非骨髓來源的細(xì)胞,可表達(dá)干細(xì)胞標(biāo)志Sca-1及早期平滑肌分化標(biāo)志SM-22α等,但不能表達(dá)SM-MHC。該群細(xì)胞可遷移進(jìn)入血管內(nèi)膜逐漸表達(dá)SM-MHC,并成為增生內(nèi)膜的主要細(xì)胞。通過流式分選技術(shù)從血管外膜將該群細(xì)胞分選后并在體外培養(yǎng)發(fā)現(xiàn)在體外經(jīng)過PDGF-BB或TGF-β誘導(dǎo)可表達(dá)SM-MHC。再體外培養(yǎng)純化的平滑肌祖細(xì)胞可從外膜遷移入內(nèi)膜并加重移植物血管病變的內(nèi)膜增生。 結(jié)論：移植后受體的中非骨髓細(xì)胞來源的平滑肌祖細(xì)胞可遷移入血管內(nèi)膜并分化為表達(dá)SM-MHC的平滑肌樣細(xì)胞,從而促進(jìn)內(nèi)膜增生病變。 第二部分miR-155可調(diào)控單核細(xì)胞趨化蛋白-1(MCP-1)濃度梯度誘導(dǎo)外膜血管平滑肌祖細(xì)胞向內(nèi)膜定向遷移 目的：了解miR-155在移植物血管病變中的作用以及對骨髓來源的單核細(xì)胞和平滑肌祖細(xì)胞的功能影響。 方法：將C57BL/6背景的miR-155基因敲除鼠和野生型C57BL/6小鼠進(jìn)行交叉骨髓移植后再作為主動脈移植的受體。流式細(xì)胞儀分選出移植物血管中骨髓來源的單核細(xì)胞,使用RT-PCR方法測定各種趨化因子的變化水平,并轉(zhuǎn)染miR-155mimics和inhibitor后使用ELISA測定培養(yǎng)上清細(xì)胞因子變化情況。使用RT-PCR方法測定移植血管標(biāo)本內(nèi)膜外膜間趨化因子濃度梯度水平,并通過Transwell實驗測定在骨髓來源的單核細(xì)胞對平滑肌祖細(xì)胞遷移能力的誘導(dǎo)作用及機制。 結(jié)果：術(shù)后八周我們發(fā)現(xiàn)接受了miR-155敲除鼠骨髓細(xì)胞的小鼠內(nèi)膜增生病變最輕,而即使是miR-155敲除鼠接受了miR-155+/+骨髓細(xì)胞小鼠也發(fā)生明顯內(nèi)膜增生。miR-155可通過抑制骨髓來源單核細(xì)胞B細(xì)胞淋巴瘤因子6的表達(dá)而促進(jìn)單核細(xì)胞趨化因子-1(MCP-1)的表達(dá)從而誘導(dǎo)平滑肌祖細(xì)胞的發(fā)生定向遷移。當(dāng)骨髓來源的單核細(xì)胞中miR-155被敲除后在移植血管內(nèi)膜外膜間MCP-1濃度梯度大幅度降低以至于難以形成有效的濃度梯度。 結(jié)論：miR-155可通過調(diào)控骨髓來源單核細(xì)胞功能分泌MCP-1而改變內(nèi)膜外膜之間濃度梯度,促進(jìn)平滑肌祖細(xì)胞向內(nèi)膜遷移。 第三部分TGF-βI型受體磷酸激酶抑制劑(SD-208)可抑制平滑肌祖細(xì)胞的增殖和遷移能力 目的：評價TGF-βI型受體磷酸化酶抑制劑(SD-208)能否抑制內(nèi)膜平滑肌樣細(xì)胞的增殖及遷移能力而減輕移植物血管病變。 方法：BALB/c小鼠主動脈移植到C57BL/6小鼠腹主動脈,術(shù)后每天給予40或60mg/kg的SD-208灌胃,術(shù)后1、2、4、6、8周取出移植血管分析內(nèi)膜增生程度并通過免疫組化方法分析TGF-β/Smad3通路各蛋白表達(dá)情況。體外培養(yǎng)中層平滑肌細(xì)胞及內(nèi)膜平滑肌樣細(xì)胞使用TGF-β and SD-208對于兩種細(xì)胞進(jìn)行干預(yù),了解增殖、遷移能力的異同。 結(jié)果：在給予40及60mg/kg等不同劑量的SD-208治療后,移植血管的內(nèi)膜增生程度分別比對照組下降了32%和48%。SD-208可以抑制TGF-p對于內(nèi)膜平滑肌樣細(xì)胞增殖和遷移能力的促進(jìn)作用而對于其對于中層平滑肌細(xì)胞的抑制作用并沒有明顯影響。SD-208可明顯減低結(jié)締組織生長因子的表達(dá)水平。在內(nèi)膜平滑肌樣細(xì)胞中Smad3的水平明顯高于中層平滑肌細(xì)胞 結(jié)論：SD-208可有效抑制內(nèi)膜平滑肌樣細(xì)胞的增殖和遷移能力,減少細(xì)胞外基質(zhì)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 .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R654.3

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1 馬寧濤;高平進(jìn);;平滑肌祖細(xì)胞研究進(jìn)展[J];生理科學(xué)進(jìn)展;2008年01期

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