【摘要】：背景:陰道平滑肌細(xì)胞原代培養(yǎng)主要有兩種方法:酶消化法和組織塊貼壁法。前者培養(yǎng)所需時(shí)間短,產(chǎn)量高,但所需組織量較大,且試驗(yàn)中污染機(jī)會(huì)大,最佳消化時(shí)間不易把握;后者雖方法簡(jiǎn)便、有效,但培養(yǎng)所需時(shí)間較長(zhǎng)。目的:觀察組織塊+酶消化法原代培養(yǎng)兔陰道平滑肌細(xì)胞時(shí)間,并與組織塊法比較。設(shè)計(jì)、時(shí)間及地點(diǎn):體外觀察實(shí)驗(yàn),于2005-02/2006-02在南昌大學(xué)第一附屬醫(yī)院燒傷研究所及動(dòng)物實(shí)驗(yàn)室完成。材料:四五個(gè)月齡雌性新西蘭大白兔,體質(zhì)量2.0～2.5kg。方法:①組織塊法原代培養(yǎng)陰道平滑肌細(xì)胞:將修剪好的1mm×1mm×1mm組織塊均勻地接種于培養(yǎng)皿中,然后放入37℃恒溫培養(yǎng)箱中靜置培養(yǎng)3.0～4.0h,待組織塊貼壁牢固后,再補(bǔ)加含體積分?jǐn)?shù)為0.1胎牛血清的DMEM培養(yǎng)液,使組織塊浸于培養(yǎng)液中,37℃恒溫靜置培養(yǎng)3.0～4.0d,待細(xì)胞游出后換液,大約每3天換液一次。②組織塊+酶消化法原代培養(yǎng)陰道平滑肌細(xì)胞:將組織塊用0.1%膠原酶Ⅳ在37℃培養(yǎng)箱中消化0.5～1.0h,至組織塊邊緣變毛時(shí)中止消化,然后將消化好的組織塊接種于培養(yǎng)皿上,其余步驟同組織塊法。③傳代培養(yǎng):第1次傳代時(shí)機(jī)應(yīng)根據(jù)細(xì)胞生長(zhǎng)具體情況而定,在細(xì)胞達(dá)50%～60%融合時(shí)即進(jìn)行傳代培養(yǎng)。第2次以后傳代,在細(xì)胞生長(zhǎng)達(dá)80%融合時(shí)進(jìn)行傳代培養(yǎng)。主要觀察指標(biāo):①原代培養(yǎng)所需時(shí)間。②第3代陰道平滑肌細(xì)胞表面結(jié)構(gòu)和超微結(jié)構(gòu)。結(jié)果:組織塊+酶消化法原代培養(yǎng)細(xì)胞萌出時(shí)間較組織塊法早1.0～2.0d,細(xì)胞融合時(shí)間較組織塊法早3.0～4.0d,細(xì)胞可穩(wěn)定傳5～6代。倒置顯微鏡下觀察兩種方法所培養(yǎng)細(xì)胞呈梭形或多角形,融合時(shí)部分區(qū)域細(xì)胞出現(xiàn)平滑肌細(xì)胞特征性生長(zhǎng)表現(xiàn)"峰-谷"狀結(jié)構(gòu)。透射電鏡下觀察,第3代陰道平滑肌細(xì)胞核呈鋸齒狀,胞漿內(nèi)含平滑肌細(xì)胞特征性結(jié)構(gòu)肌絲和密體,含有大量高爾基體,粗面內(nèi)質(zhì)網(wǎng)擴(kuò)張,游離核糖體豐富,提示獲得合成表型陰道平滑肌細(xì)胞比例大。結(jié)論:組織塊+酶消化法原代培養(yǎng)周期較短,需要注意的是在取材、分離整個(gè)操作過(guò)程中需保持陰道組織塊濕潤(rùn);膠原酶消化組織塊時(shí)間不應(yīng)過(guò)長(zhǎng),以組織塊周邊呈現(xiàn)毛須狀為度;此外應(yīng)在靜置狀態(tài)下培養(yǎng)。
[Abstract]:Background: there are two methods in primary culture of vaginal smooth muscle cells: enzyme digestion and tissue mass adherence. The former has the advantages of short time, high yield, large amount of tissue, large pollution opportunity in the experiment, and the best digestion time is difficult to grasp. The latter method is simple and effective, but it takes a long time to culture. Aim: to observe the time of primary culture of rabbit vaginal smooth muscle cells by tissue mass enzyme digestion and compare with tissue block method. Design, time and place: in vitro observation experiment was completed in the Institute of Burn and Animal Laboratory, first affiliated Hospital of Nanchang University, February 2006, 2005. Materials: female New Zealand white rabbits of four or five months of age, with a body mass of 2. 0 ~ 2. 5 kg. Methods: 1 Primary culture of vaginal smooth muscle cells by tissue block method: the trimmed 1mm 脳 1mm 脳 1mm tissue blocks were evenly inoculated into the culture dish, and then cultured in a 37 鈩，

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