發(fā)布時間：2018-11-06 08:54

【摘要】：目的:觀察降鈣素基因相關(guān)肽在體外培養(yǎng)細胞中的作用,探究降鈣素基因相關(guān)肽對大鼠胃平滑肌的調(diào)控作用和機制。方法:幼年SD大鼠胃,采用組織碎塊貼瓶底和消化酶消化兩種方法培養(yǎng)SD大鼠胃平滑肌細胞,成功提取胃平滑肌細胞(smooth muscle cell,SMC)并用高糖DMEM營養(yǎng)液(含20%血清)培養(yǎng)。分離成功的平滑肌細胞利用可發(fā)生特異反應(yīng)的α-actin細胞組化進行鑒別。實驗分為:CGRPI、CGRPII、CGRPIII、CGRP8-37+CGRP組、空白對照共5組,加入相對應(yīng)的試劑。利用電腦數(shù)據(jù)分析系統(tǒng)測量平滑肌細胞加藥前后長短;用鈣熒光標(biāo)志劑測測胞內(nèi)的Ca2+濃度;Celltiter-Glo(?)組合試劑測定細胞內(nèi)ATP含量。結(jié)果:貼塊法和酶解法都能提取細胞,酶解法所提細胞所用時間短,但雜質(zhì)較多,貼塊法雖然時間相對較長但細胞純度相對較高。成功分離SD大鼠胃平滑肌細胞,細胞呈現(xiàn)梭子狀或是無規(guī)則圖形,細胞核處在細胞中間。免疫組化鑒定能夠看到平滑肌細胞成無規(guī)則形,細胞漿呈很淺的棕黃色,細胞核呈淡藍色,表明提取的正是實驗所需的胃平滑肌細胞。CGRP在各個劑量時都能夠降低胃腸平滑肌胞漿里Ca2+含量,并在一定范圍內(nèi)隨CGRP劑量增加降低Ca2+含量作用增大。加入CGRP后和對照組相比能降低平滑肌細胞所攜帶的ATP,并且隨著CGRP濃度的增加ATP的含量改變也增大(P0.05)。結(jié)論:應(yīng)用組織碎塊貼瓶底法得到的胃平滑肌細胞,通過繼續(xù)培養(yǎng)、時差貼瓶底等可以得到所需的鼠胃SMC。由實驗可以看出CGRP能松弛胃平滑肌,降低細胞內(nèi)鈣離子濃度及ATP含量,提示鈣離子濃度變化可能參與了其松弛平滑肌細胞的機制;CGRP可能通過改變胃平滑肌細胞動能來抑制胃SM運動。
[Abstract]:Aim: to investigate the effect of calcitonin gene-related peptide (CGRP) on rat gastric smooth muscle in vitro. Methods: gastric smooth muscle cells (GSMCs) of SD rats were cultured by tissue fragmentation and digestive enzyme digestion. Gastric smooth muscle cells (smooth muscle cell,SMC) were extracted successfully and cultured with high glucose DMEM nutrient solution (containing 20% serum). The isolated smooth muscle cells were identified by histochemistry of 偽-actin cells that could react specifically. The experiment was divided into CGRPI,CGRPII,CGRPIII,CGRP8-37 CGRP group and blank control group. Computer data analysis system was used to measure the length of smooth muscle cells before and after drug addition; calcium fluorescent marker was used to measure intracellular Ca2 concentration; Celltiter-Glo (?) The content of ATP in cells was determined by combined reagent. Results: the cells could be extracted by the method of sticking and enzymatic hydrolysis. The time of the cells extracted by enzymatic hydrolysis was short, but the impurity was more. Although the time of the method was relatively long, the purity of the cells was relatively high. Gastric smooth muscle cells of SD rats were isolated successfully. The cells showed spindle shape or irregular pattern and the nucleus was in the middle of the cells. Immunohistochemical staining showed that smooth muscle cells were irregular in shape, the cytoplasm was very light brown, and the nucleus was light blue. The results showed that the gastric smooth muscle cells (GSMCs) were extracted. CGRP could decrease the content of Ca2 in the cytoplasm of gastrointestinal smooth muscle at every dose and increase the content of Ca2 with the increase of CGRP dose in a certain range. Compared with the control group, the addition of CGRP decreased the ATP, carried by smooth muscle cells and increased the content of ATP with the increase of CGRP concentration (P0.05). Conclusion: the gastric smooth muscle cells obtained by using tissue fragments and flask bottom method can be used to obtain the required gastric SMC. through continuous culture and jet-lag sticking to the bottom of the bottle. It can be seen from the experiment that CGRP can relax gastric smooth muscle and decrease intracellular calcium ion concentration and ATP content, suggesting that the change of calcium ion concentration may be involved in the mechanism of relaxation smooth muscle cells. CGRP may inhibit gastric SM motility by changing the kinetic energy of gastric smooth muscle cells.
【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R363

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