本文關鍵詞：眼瞼基底細胞癌相關成纖維細胞的分離培養(yǎng)及生物學特性研究　出處：《川北醫(yī)學院》2016年碩士論文　論文類型：學位論文

  更多相關文章： 眼瞼基底細胞癌 癌相關成纖維細胞 正常成纖維細胞 分離 培養(yǎng) 生物學特性

【摘要】：目的:對眼瞼基底細胞癌相關成纖維細胞(CAFs)及眼瞼正常成纖維細胞(NFs)進行分離、培養(yǎng)、純化、鑒定,初步研究CAFs與NFs的細胞形態(tài)、生長增殖及骨膜蛋白(PN或POSTN)表達等生物學特性的差異。方法:(1)用組織塊貼壁法培養(yǎng)獲得原代眼瞼基底細胞癌CAFs和眼瞼正常NFs,通過胰酶差速消化法和反復傳代培養(yǎng)法進行細胞的分離、純化;(2)通過倒置相差顯微鏡觀察CAFs和NFs細胞形態(tài),應用免疫細胞化學染色法檢測細胞表達的特異性蛋白,對CAFs和NFs進行鑒定;(3)采用MTT比色法對連續(xù)培養(yǎng)7天的細胞數(shù)量進行檢測并繪制生長曲線,通過酶聯(lián)免疫吸附試驗(ELISA)法檢測CAFs與NFs細胞培養(yǎng)上清液中PN的含量,應用免疫熒光細胞化學染色方法檢測CAFs與NFs中PN的表達。結果:(1)成功分離出了純化的眼瞼CAFs和NFs;(2)眼瞼CAFs呈長梭形,胞質突減少,胞漿內(nèi)可見較多顆粒,細胞大小不一,生長較快,排列紊亂,有重疊生長現(xiàn)象;眼瞼CAFs和NFs的細胞免疫化學染色,前者波形蛋白(VIM)、α平滑肌肌動蛋白(α-SMA)、成纖維細胞活化蛋白(FAP)呈陽性,細胞角蛋白(CK)呈陰性,后者僅VIM呈陽性,CK、α-SMA、FAP均呈陰性。(3)與NFs相比,眼瞼CAFs生長增殖較快,倍增時間較短;ELISA法測定CAFs與NFs細胞培養(yǎng)上清液中PN的含量,分別為((9.26±2.35)ng/ml)和((2.57±0.41)ng/ml);細胞免疫熒光化學染色檢測眼瞼CAFs中PN表達強于NFs,差異具有統(tǒng)計學意義(P0.05)。結論:與NFs相比,眼瞼基底細胞癌中CAFs在形態(tài)結構、生長增殖、骨膜蛋白表達等生物學特征存在顯著差異,提示眼瞼基底細胞癌細胞微環(huán)境的變化導致NFs生物學特性和功能的改變而形成了CAFs,同時與眼瞼基底細胞癌細胞相互作用,促進或抑制眼瞼基底細胞癌的發(fā)生發(fā)展。
[Abstract]:Objective: to isolate, culture, purify and identify the basal cell carcinoma associated fibroblasts (CAFs) and normal eyelid fibroblasts (NFS). The cell morphology of CAFs and NFs was studied. Methods CAFs and normal eyelid NFs were obtained from primary eyelid basal cell carcinoma (BCC) and normal eyelid (NFs). The cells were separated and purified by trypsin differential digestion and repeated passage culture. (2) the morphology of CAFs and NFs cells were observed by inverted phase contrast microscope, and the specific proteins expressed by the cells were detected by immunocytochemical staining. CAFs and NFs were identified. MTT colorimetry was used to detect the number of cells cultured for 7 days and to draw the growth curve. The content of PN in the supernatant of CAFs and NFs cells was detected by Elisa. The expression of PN in CAFs and NFs was detected by immunofluorescence cytochemical staining. (2) CAFs of eyelid is fusiform, cytoplasmic process is decreased, more granules can be seen in the cytoplasm, cell size is different, growth is faster, arrangement is disordered, and there is overlapping growth phenomenon; The vimentin vimentin (VIMA) and 偽 smooth muscle actin (偽 -SMAA) were positive in eyelid CAFs and NFs. Cytokeratin (CK) was negative, only VIM was positive, 偽 -SMA-FAP was negative. (3) compared with NFs, CAFs of eyelid grew and proliferated faster and the doubling time was shorter. The content of PN in supernatant of CAFs and NFs cells was determined by ELISA method. They were 9.26 鹵2.35 ng / ml and 2.57 鹵0.41 ng / ml, respectively. The expression of PN in eyelid CAFs was stronger than that in NFS by immunofluorescence staining, and the difference was statistically significant (P 0.05). Conclusion: compared with NFs. There were significant differences in the biological characteristics of CAFs in eyelid basal cell carcinoma (BCC), such as morphology, growth and proliferation, and expression of periosteal protein. It is suggested that the changes of microenvironment of eyelid basal cell carcinoma cells lead to the change of biological characteristics and function of NFs, which results in the formation of CAFs, and the interaction with BCC cells. Promote or inhibit the development of basal cell carcinoma of eyelid.
【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R739.71

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