發(fā)布時間：2018-06-08 22:00

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 陰道脫細(xì)胞基質(zhì)�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探索應(yīng)用骨髓間充質(zhì)干細(xì)胞(BMSCs)復(fù)合豬陰道脫細(xì)胞基質(zhì)(AVM)構(gòu)建的大鼠組織工程陰道是否優(yōu)于單純應(yīng)用AVM。方法:取20日齡雄性SD大鼠分離雙側(cè)股骨及脛骨,采用全骨髓貼壁法培養(yǎng)BMSCs。取第3代BMSCs與AVM復(fù)合培養(yǎng)24h,掃描電鏡觀察細(xì)胞在支架上的生長情況。CM.dil標(biāo)記第3代細(xì)胞,熒光顯微鏡觀察染色情況及標(biāo)記效率。將第3代BMSCs以1×105/cm2的細(xì)胞密度與AVM復(fù)合培養(yǎng),分別于復(fù)合后1、3、5、7天行MTT法檢測BMSCs在AVM上的增殖情況。經(jīng)陰道全切雌性SD大鼠陰道構(gòu)建動物模型。根據(jù)構(gòu)建方式不同分為三組:(1)標(biāo)記的BMSCs復(fù)合AVM,即復(fù)合BMSCs組,(2)單純AVM構(gòu)建大鼠組織工程陰道,即單純支架組,(3)正常大鼠陰道作為對照組。分別于術(shù)后1、2、3、4、8周取材行大體形態(tài)觀察,HE、Masson染色行微觀形態(tài)學(xué)觀察,冰凍切片熒光顯微鏡下觀察陰道組織中BMSCs向上皮細(xì)胞,平滑肌細(xì)胞、神經(jīng)細(xì)胞、血管內(nèi)皮細(xì)胞的分化情況。組織浴槽法對新陰道進(jìn)行功能學(xué)評價,試驗開始前給予124 m M的高鉀溶液,若反應(yīng)不良將組織條棄掉。給予頻率(10~100 Hz),電壓30V的電刺激,觀察肌條反應(yīng)情況,再分別給予不同濃度的去氧腎上腺素(10-6~2*10-4M)、酚妥拉明(10-5~2*10-4M)、卡巴膽堿(10-6~10-4M)、阿托品(10-5~10-4M)后觀察肌條反應(yīng)。結(jié)果:全骨髓貼壁法培養(yǎng)的BMSCs具有成纖維細(xì)胞樣形態(tài),90%融合時成漩渦狀或放射狀生長。熒光顯微鏡觀察CM.dil標(biāo)記后的BMSCs,骨髓間充質(zhì)干細(xì)胞胞膜呈紅色熒光,細(xì)胞核不著色,標(biāo)記效率幾乎可達(dá)100%。掃描電鏡觀察發(fā)現(xiàn)大體觀察發(fā)現(xiàn),BMSCs牢固附著于AVM上,呈長梭形。MTT法檢測BMSCs與AVM復(fù)合培養(yǎng)后第7天細(xì)胞在支架上的細(xì)胞活性最佳。復(fù)合BMSCs組與單純支架組均形成了新生陰道,術(shù)后8周兩組新陰道均粘膜化,接近正常組織形態(tài)。HE、Masson染色后微觀形態(tài)學(xué)觀察發(fā)現(xiàn),術(shù)后1周,復(fù)合BMSCs組可見少量上皮組織,單純支架組僅可見未降解的AVM支架材料,兩組均可見較多炎癥細(xì)胞浸潤。術(shù)后2周,復(fù)合BMSCs組上皮組織覆蓋重建陰道全長,單純支架組術(shù)后3周上皮組織才達(dá)重建陰道全長,術(shù)后4周兩組上皮層細(xì)胞排列更加規(guī)則,更接近正常組織。復(fù)合BMSCs組纖維組織較單純支架組排列更加規(guī)整。術(shù)后8周復(fù)合BMSCs組可見大量規(guī)整的平滑肌纖維,單純支架組僅可見雜亂纖細(xì)的肌纖維。免疫熒光染色發(fā)現(xiàn)BMSCs可直接分化為平滑肌細(xì)胞、血管內(nèi)皮細(xì)胞、神經(jīng)細(xì)胞促進(jìn)組織的再生。BMSCs也可能通過旁分泌作用、免疫調(diào)節(jié)作用促進(jìn)組織再生。功能學(xué)檢測發(fā)現(xiàn),復(fù)合BMSCs組新陰道對藥物及電刺激反應(yīng)均優(yōu)于單純支架組。結(jié)論:復(fù)合BMSCs組與單純支架組均可成功構(gòu)建組織工程陰道,復(fù)合BMSCs組織工程陰道從形態(tài)學(xué)及功能學(xué)方面均優(yōu)于單純AVM支架組。BMSCs可通過直接分化作用促進(jìn)大鼠組織工程陰道重建,并且可能通過旁分泌作用及免疫調(diào)節(jié)作用促進(jìn)大鼠組織工程陰道重建。
[Abstract]:Objective: to explore whether the tissue engineered vagina constructed by bone marrow mesenchymal stem cell (BMSCs) combined with porcine vaginal acellular stroma AVMwas superior to AVM alone. Methods: bilateral femur and tibia were isolated from 20 day old male Sprague-Dawley rats and BMSCs were cultured by whole bone marrow adherent method. The third passage BMSCs were co-cultured with AVM for 24 hours. The growth of the cells on the scaffold was observed by scanning electron microscope. The third passage cells were labeled with CM.dil. The staining and labeling efficiency were observed by fluorescence microscope. The third generation of BMSCs were co-cultured with 1 脳 105/cm2 cell density. The proliferation of BMSCs was detected by MTT assay on the 7th day after 1 脳 105/cm2. The vaginal model of female Sprague-Dawley rats was established. According to different construction methods, BMSCs labeled with BMSCs were divided into three groups: BMSCs combined with AVM (compound BMSCs group) and simple AVM to construct rat tissue engineered vagina, that is, simple scaffold group (n = 3) normal rat vagina was used as control group. The morphology of BMSCs in vaginal tissue was observed under fluorescence microscope. The differentiation of BMSCs into epithelial cells, smooth muscle cells, nerve cells and vascular endothelial cells was observed under fluorescence microscope. The function of the new vagina was evaluated by tissue bath method. 124mm high potassium solution was given before the experiment. If the reaction was bad, the tissue strip would be discarded. The reactivity of muscle strips was observed by electrical stimulation with a frequency of 10 ~ 100 Hz and a voltage of 30 V, followed by different concentrations of noradrenaline (10 ~ (-6) ~ (-2) ~ (-4) M ~ (-1), phentoid Lemine (10 ~ (-5) ~ (2) 10 ~ (-4) M ~ (4), carbachol 10 ~ (-6) ~ (10 ~ (-4) M) and atropine (10 ~ (-5) 10 ~ (-4) M). Results: BMSCs cultured by whole bone marrow adherent method had 90% fibroblast-like morphology. The cell membrane of bone marrow mesenchymal stem cells was red fluorescent, the nucleus was not stained, and the labeling efficiency was almost 100%. Scanning electron microscopy (SEM) showed that BMSCs were firmly attached to AVM, and the cell activity on the scaffold was the best on the 7th day after co-culture of BMSCs and AVM. The new vagina was formed in both the composite BMSCs group and the simple stent group. At 8 weeks after operation, the new vagina became mucous, close to the normal tissue morphology. The microscopic morphological observation showed that a small amount of epithelial tissue was found in the composite BMSCs group at 1 week after operation. Only non-degradable AVM scaffolds were found in the pure stent group, and more inflammatory cell infiltration was observed in both groups. At 2 weeks after operation, the epithelium of composite BMSCs group was covered with the whole length of the reconstructed vagina, while that of the simple stent group reached the full length of the reconstructed vagina 3 weeks after operation, and the arrangement of epithelial cells in the two groups was more regular and closer to the normal tissue 4 weeks after operation. The arrangement of fiber tissue in composite BMSCs group was more regular than that in simple scaffold group. 8 weeks after operation, a large number of regular smooth muscle fibers were observed in the combined BMSCs group, but only in the simple scaffold group. Immunofluorescence staining showed that BMSCs could differentiate directly into smooth muscle cells, vascular endothelial cells, nerve cells, promote tissue regeneration. BMSCs may also promote tissue regeneration through paracrine and immunomodulation. The results of functional examination showed that the new vagina response to drugs and electrical stimulation in BMSCs group was better than that in stent group. Conclusion: the tissue engineered vagina can be successfully constructed in the combined BMSCs group and the simple scaffold group. The compound BMSCs tissue engineered vagina is superior to the AVM scaffold group in morphology and function. BMSCs can promote the reconstruction of rat tissue engineering vagina through direct differentiation. The paracrine and immunomodulatory effects may promote the reconstruction of tissue engineered vagina in rats.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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