【摘要】：目的：探討螺旋形聚乳酸支架皮下誘導(dǎo)結(jié)締組織薄膜形成的規(guī)律及包埋時(shí)間對(duì)尿路上皮細(xì)胞增殖的影響，最終體外構(gòu)建出具有一定生物活性的組織工程輸尿管。方法： Wistar大鼠膀胱活檢獲得泌尿道粘膜標(biāo)本，酶消化法原代分離尿路上皮細(xì)胞。用分子量為1.5×10~5Da的聚乳酸制備螺旋形輸尿管內(nèi)支架和片狀薄膜，前者用于探討皮下誘導(dǎo)結(jié)締組織薄膜形成的規(guī)律，后者用于探討包埋時(shí)間對(duì)尿路上皮細(xì)胞增殖的影響。將螺旋形聚乳酸輸尿管內(nèi)支架和片狀聚乳酸薄膜包埋到Wistar大鼠皮下，在不同的時(shí)間點(diǎn)（1、2、3周）將螺旋形聚乳酸輸尿管內(nèi)支架和片狀聚乳酸薄膜連同表面結(jié)締組織一起取回，經(jīng)脫細(xì)胞處理清除結(jié)締組織層中的細(xì)胞成分。將體外擴(kuò)增的尿路上皮細(xì)胞按4×10~4cells/cm~2的密度接種到螺旋形聚乳酸輸尿管內(nèi)支架和片狀聚乳酸薄膜表面的脫細(xì)胞結(jié)締組織層，檢測(cè)尿路上皮細(xì)胞在結(jié)締組織層的附著、增殖情況。結(jié)果：成功原代分離膀胱尿路上皮細(xì)胞，經(jīng)角朊上皮無(wú)血清培養(yǎng)基（DSFM）培養(yǎng)后免疫組織化學(xué)染色證實(shí)為純化的尿路上皮細(xì)胞；細(xì)胞排列為典型的鋪路石樣外觀，4代以前的細(xì)胞狀態(tài)良好，可作為組織工程輸尿管構(gòu)建的理想種子細(xì)胞；包埋1周，在螺旋形聚乳酸輸尿管內(nèi)支架表面形成了一層菲薄的組織薄膜，炎癥反應(yīng)輕微，可見(jiàn)少量多型核白細(xì)胞存在，無(wú)新生血管存在；包埋2周，在螺旋形聚乳酸輸尿管內(nèi)支架表面可見(jiàn)薄層結(jié)締組織形成，在結(jié)締組織周邊可見(jiàn)少量毛細(xì)血管存在；包埋3周，螺旋形聚乳酸輸尿管內(nèi)支架表面結(jié)締組織中有纖維增生表現(xiàn)，可見(jiàn)大量毛細(xì)血管存在，炎癥反應(yīng)完全消失。各包埋時(shí)間組，，結(jié)締組織經(jīng)脫細(xì)胞處理后可見(jiàn)大量膠原組織殘留，經(jīng)免疫組織化學(xué)鑒定其內(nèi)含有豐富的Ⅰ型膠原蛋白、Ⅳ型膠原蛋白、層粘連蛋白、纖維連接蛋白，這些蛋白組成與尿路上皮基底層主要成分相近。在各包埋時(shí)間組，掃描電子顯微鏡觀察發(fā)現(xiàn)脫細(xì)胞結(jié)締組織表面均呈三維立體結(jié)構(gòu)；與包埋2、3周組相比，包埋1周組的脫細(xì)胞結(jié)締組織較薄，部分區(qū)域暴露出內(nèi)層的聚乳酸支架部分；脫細(xì)胞結(jié)締組織種植尿路上皮細(xì)胞3天后，尿路上皮細(xì)胞附著良好，細(xì)胞周邊有偽足樣結(jié)構(gòu)伸出，并沿結(jié)締組織表面延伸。MTT結(jié)果顯示，在不同包埋時(shí)間組，尿路上皮細(xì)胞在脫細(xì)胞結(jié)締組織層均能連續(xù)增殖。體外孵育5、7天，與包埋1周組相比，尿路上皮細(xì)胞在包埋2、3周的脫細(xì)胞結(jié)締組織層中具有更高的增殖活性（p0.05）。結(jié)論：體外擴(kuò)增的尿路上皮細(xì)胞可種植于體內(nèi)誘導(dǎo)形成的脫細(xì)胞結(jié)締組織上構(gòu)建出具有一定生物活性的組織工程輸尿管。綜合結(jié)締組織薄膜脫細(xì)胞前后組織病理學(xué)檢測(cè)結(jié)果及尿路上皮細(xì)胞在脫細(xì)胞結(jié)締組織層中的增殖情況，我們認(rèn)為包埋2、3周的脫細(xì)胞結(jié)締組織是體外構(gòu)建組織工程輸尿管的良好介質(zhì)；該研究結(jié)果可為皮下誘導(dǎo)形成結(jié)締組織薄膜、種植自身尿路上皮細(xì)胞后構(gòu)建組織工程輸尿管奠定實(shí)驗(yàn)基礎(chǔ)。盡管如此，該組織工程輸尿管用于輸尿管損傷修復(fù)、重建的有效性和安全性，仍待進(jìn)一步動(dòng)物實(shí)驗(yàn)研究證實(shí)。
[Abstract]:Objective: To investigate the regularity of the formation of connective tissue film under the subcutaneous induction of spiral polylactic acid stent and the effect of embedding time on the proliferation of urinary tract epithelial cells. Finally, a tissue engineering ureter with certain biological activity was constructed in vitro. Methods: urinary tract mucosa specimens were obtained by bladder biopsy in Wistar rats, and the original urinary tract was separated by enzyme digestion. Epithelial cells. A spiral ureteral stent and flake thin film were prepared with a molecular weight of 1.5 * 10~5Da polylactic acid. The former was used to investigate the formation of subcutaneous induced connective tissue film. The latter was used to explore the effect of embedding time on the proliferation of urinary tract epithelial cells. The spiral polylactic ureteral stent and flaky polylactic acid film were retrieved together with the surface connective tissue at different time points (1,2,3 weeks) to remove the cell components in the connective tissue layer at different time points (Wistar weeks). The cells expanded in vitro were inoculated into the spiral shape at 4 x 10~4cells/cm~2 density. Polylactic ureteral stent and the decellularized connective tissue layer on the surface of flaky polylactic acid film to detect the attachment and proliferation of urinary tract epithelial cells in connective tissue layer. Results: successful primary separation of bladder urothelial cells, and immuno histochemical staining of the serum-free culture medium of keratinocyte (DSFM) proved to be the purified urinary tract. Epithelial cells; cells arranged as a typical paving stone appearance. The 4 generation of cells were in good condition and could be used as ideal seed cells for the construction of the tissue engineered ureter. A thin layer of thin tissue film was formed on the surface of the spiral polylactic ureteral stent for 1 weeks, and a slight inflammatory reaction was found, and a small amount of polymorphonuclear leukocytes were found. No neovascularization existed; 2 weeks of embedding, a thin connective tissue formed on the surface of the spiral polylactic ureteral stent, and a small amount of capillaries were found around the connective tissue; for 3 weeks, fibrous proliferation in the surface connective tissue of the spiral polylactic acid ureteral stent showed a large number of capillary and inflammatory reactions. Complete disappearance. A large amount of collagen tissue remained in the connective tissue after acellular treatment. It was identified by immuno histochemistry as a rich type of collagen type I, type IV collagen, laminin, fibronectin, and these proteins were similar to the main components in the bottom of the urinary tract. The scanning electron microscope showed that the surface of the acellular connective tissue was three-dimensional structure. Compared with the embedded 2,3 week group, the decellated connective tissue was thinner in the 1 week group, and the inner layer of the polylactic acid scaffold was exposed in some areas. After 3 days after the acellular connective tissue planted the urinary tract cells, the urinary tract epithelial cells attached well and fine. The perimeter of the cell was protruded and extended along the connective tissue surface.MTT results showed that the urinary tract epithelial cells were able to proliferate continuously in the acellular connective tissue layer at different embedding time groups. In vitro incubation for 5,7 days, the urinary tract epithelial cells had a higher proliferation activity in the 1 week embedded connective tissue layer embedded in the embedded 2,3 week group. P0.05. Conclusion: in vitro expanded urinary tract epithelial cells can be planted in the acellular connective tissue induced by the body to construct a tissue engineered ureter with certain biological activity. The histological examination of the tissue film before and after the dehydration of the connective tissue film and the proliferation of the urinary tract epithelial cells in the decellular connective tissue layer In addition, we believe that the acellular connective tissue embedded in 2,3 weeks is a good medium for constructing the tissue engineered ureter in vitro. The results can provide an experimental basis for subcutaneous induction of connective tissue film and the construction of tissue engineered ureter after the cultivation of its own urinary tract epithelial cells. The effectiveness and safety of wound repair and reconstruction remain to be confirmed by animal experiments.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 盧洪凱;任安吉;孫曉璐;高恩江;于志勇;閆波;;復(fù)合雪旺細(xì)胞的豬小腸粘膜下層對(duì)大鼠海綿體神經(jīng)損傷勃起功能恢復(fù)的實(shí)驗(yàn)研究[J];中華男科學(xué)雜志;2010年09期

2 閆國(guó)和;羊膜基質(zhì)在組織修復(fù)中的應(yīng)用研究進(jìn)展[J];生物醫(yī)學(xué)工程學(xué)雜志;2002年04期

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