本文選題：貼壁培養(yǎng)法　切入點(diǎn)：酶消化法　出處：《第四軍醫(yī)大學(xué)》2010年碩士論文

【摘要】：1.目的 本研究的目的是通過聯(lián)合兩種常用的細(xì)胞培養(yǎng)方法(酶消化培養(yǎng)法和組織貼壁培養(yǎng)法),建立了一個(gè)體外的蝸軸螺旋動(dòng)脈平滑肌細(xì)胞原代培養(yǎng)的模型。 2.方法 2.1用于進(jìn)行實(shí)驗(yàn)的的蝸軸螺旋動(dòng)脈的平滑肌來自于豚鼠。將蝸軸螺旋動(dòng)脈血管組織切碎并且放入37度二氧化碳培養(yǎng)箱中在溶液濃度為0.1%胰蛋白酶溶液中消化20分鐘。接著,將這些消化后的血管組織塊在35-mm的培養(yǎng)皿中貼壁,組織塊中間間隔3-5mm。 2.2聯(lián)合運(yùn)用這兩種細(xì)胞培養(yǎng)方法,我們發(fā)現(xiàn)其中混雜的細(xì)胞幾乎都為成纖維細(xì)胞,所以如何去除成纖維細(xì)胞是本實(shí)驗(yàn)需要克服的問題。經(jīng)過查閱文獻(xiàn)和多次的實(shí)踐,我們將蝸軸螺旋動(dòng)脈血管平滑肌細(xì)胞原代培養(yǎng)過程中混雜的成纖維細(xì)胞通過其與血管平滑肌細(xì)胞不同的貼壁能力和成纖維細(xì)胞對(duì)無血清的培養(yǎng)液不耐受,因?yàn)榭稍谠趥鞔臅r(shí)候被去除。當(dāng)35mm培養(yǎng)皿中細(xì)胞長(zhǎng)滿80%-90%的時(shí)候,就可以進(jìn)行傳代。在傳代過程中應(yīng)用上述的去除成纖維細(xì)胞的方法。我們將細(xì)胞懸液加入到35mm的培養(yǎng)皿中,15分鐘后,吸出液體。將吸出的液體加入另外一個(gè)35mm的培養(yǎng)皿中,靜置15分鐘。吸出液體并加入到第三個(gè)35mm的培養(yǎng)皿中。收集第二和第三個(gè)培養(yǎng)皿繼續(xù)培養(yǎng)。反復(fù)應(yīng)用純化的程序,可以在第三代得到純的并且活性好的蝸軸螺旋動(dòng)脈平滑肌細(xì)胞。 3.結(jié)果 3.1通過倒置顯微鏡的觀察,我們發(fā)現(xiàn)在7-10天后,可以見到有細(xì)胞從組織塊中長(zhǎng)出來。大約在3周左右,這些細(xì)胞可長(zhǎng)滿培養(yǎng)皿,大約在細(xì)胞長(zhǎng)滿培養(yǎng)皿80%-90%時(shí),可以進(jìn)行傳代。通過方法中介紹的血管平滑肌細(xì)胞純化的方法,在第三代培養(yǎng)的血管平滑肌細(xì)胞中,純的并且活性好的細(xì)胞可以被獲得。 3.2經(jīng)過形態(tài)學(xué),免疫熒光化學(xué)對(duì)于上述方法得到的蝸軸螺旋動(dòng)脈血管血管平滑肌細(xì)胞進(jìn)行了鑒別。這些細(xì)胞呈現(xiàn)梭形的細(xì)胞形態(tài),每個(gè)細(xì)胞含有1-2個(gè)核。結(jié)果顯示了典型的平滑肌的細(xì)胞的特點(diǎn):形態(tài)學(xué)顯示了平滑肌細(xì)胞在細(xì)胞密度大的時(shí)候出現(xiàn)的典型的“峰-谷”樣的生長(zhǎng)形態(tài)。免疫熒光化學(xué)顯示這些原代培養(yǎng)的蝸軸螺旋動(dòng)脈血管平滑肌細(xì)胞表達(dá)平滑肌細(xì)胞的特異性標(biāo)志物(α-SM- actin和myosin)。 3.3透射電鏡結(jié)果顯示的培養(yǎng)的蝸軸螺旋動(dòng)脈平滑肌細(xì)胞中具有密斑,密體,肌絲這些平滑肌細(xì)胞的標(biāo)志性結(jié)構(gòu)。 4.結(jié)論 本文通過聯(lián)合兩種常見的細(xì)胞培養(yǎng)方法,建立了一種有效并且簡(jiǎn)單的細(xì)胞培養(yǎng)方法。應(yīng)用這種原代培養(yǎng)的方法,大量純的并且活性良好的蝸軸螺旋動(dòng)脈血管平滑肌細(xì)胞可以在原代培養(yǎng)第三代的時(shí)候得到。這些血管平滑肌細(xì)胞是一個(gè)很好的研究血管平滑肌細(xì)胞在內(nèi)耳循環(huán)紊亂過程生理功能的一個(gè)體外模型。除此之外,這些活性良好的平滑肌細(xì)胞還可以是一些作用于血管平滑肌藥物評(píng)價(jià)的體外模型。
[Abstract]:1. Purpose. The aim of this study was to establish a primary culture model of cochlear spiral artery smooth muscle cells in vitro by combining two common cell culture methods (enzyme digestion culture and tissue adherence culture). 2. Methodology. 2.1 the smooth muscle of the cochlear axial spiral artery used for the experiment comes from the guinea pig. The vascular tissue of the cochlear axial spiral artery is chopped and digested in a 37 degree carbon dioxide incubator in a 0.1% trypsin solution for 20 minutes. Then, These digested vascular tissue blocks were adhered to the 35-mm dish with an interval of 3 to 5 mm. 2.2 in combination with these two methods of cell culture, we found that almost all of the mixed cells were fibroblasts, so how to remove fibroblasts is a problem to overcome in this experiment. In the primary culture of vascular smooth muscle cells of cochlear axis spiral artery, the mixed fibroblasts were treated by their different adherent ability from vascular smooth muscle cells and the intolerance of fibroblasts to serum-free culture medium. Because it can be removed during passage. When the cells in the 35mm dish are 80 to 90 percent longer, We put the cell suspensions in the culture dish of 35mm for 15 minutes, and then we suck out the liquid, and we add the liquid to another dish of 35mm. Sit still for 15 minutes. Suck out the liquid and add it to the third 35mm dish. Collect the second and third dishes and continue the culture. Repeat the purification process. Pure and active smooth muscle cells of the cochlear axial spiral artery can be obtained in the third generation. 3. Results. 3.1 from the observation of the inverted microscope, we found that after 7-10 days, cells could be seen growing out of the tissue mass. In about 3 weeks, these cells could be filled with petri dishes, about 80 to 90 percent of the time when the cells were full of petri dishes. In the third passage of vascular smooth muscle cells, pure and active cells can be obtained. 3.2 morphologically, immunofluorescence was used to identify vascular smooth muscle cells of the cochlear axial spiral artery obtained by the above method. Each cell contains 1-2 nuclei. The results show the characteristics of typical smooth muscle cells: morphology shows typical "peak-valley" growth patterns of smooth muscle cells at high cell density. Immunofluorescence. Chemical analysis showed that these primary cultured vascular smooth muscle cells of the cochlear spiral artery expressed specific markers of smooth muscle cells (偽 -SM- actin and myosinine). 3.3 the results of transmission electron microscope showed that the cultured smooth muscle cells of the cochlear axial spiral artery had the iconic structures of dense spots, dense bodies and myofilms. 4. Conclusions. In this paper, an effective and simple cell culture method was established by combining two common cell culture methods. A large number of pure and active vascular smooth muscle cells of spiral cochlear artery can be obtained in the third generation of primary culture. These vascular smooth muscle cells are a good study of vascular smooth muscle cells in the inner ear circulation. An in vitro model of the physiological function of chaotic processes. These active smooth muscle cells may also be in vitro models for the evaluation of vascular smooth muscle drugs.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王燁;米文娟;韓宇;鐘翠萍;邱建華;喬莉;;蝸軸螺旋動(dòng)脈平滑肌的培養(yǎng)及其鑒定[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2010年09期

2 孫建軍;刁明芳;;急性聽力損失的診斷與治療[J];中華耳鼻咽喉頭頸外科雜志;2007年11期

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