【作者】 薛娟；

【导师】 段斐；

【作者基本信息】 河北大学 ， 药理学， 2014， 硕士

【摘要】 目的：通过体外培养MRC-5细胞，观察不同浓度盐酸川芎嗪注射液对MRC-5细胞增殖的影响；进一步观察不同浓度盐酸川芎嗪作用不同时间后，MRC-5细胞胶原蛋白表达的变化，为中药在肺纤维化的防治方面提供新的理论依据和思路。方法：1．体外培养人胚肺成纤维细胞系MRC-5细胞，用不同浓度盐酸川芎嗪进行处理，分为①正常对照组，②川芎嗪1×10-3mg/ml组③川芎嗪1×10-2mg/ml组④川芎嗪1×10-1mg/ml组⑤川芎嗪1mg/ml，在37℃、5%CO2培养箱中分别培养24h、48h、72h，首先镜下观察细胞的形态变化，再采用MTT法检测不同浓度川芎嗪作用不同时间后，MRC-5细胞在双波长（490nm、630nm）的吸光度A值，通过比较A值做时效、量效图表，增殖抑制率图表，比较不同浓度不同时间盐酸川芎嗪对MRC-5细胞增殖的影响。2．体外培养MRC-5细胞，分为①正常对照组、②地塞米松组（5×10-2mg/ml）③川芎嗪1×10-3mg/ml组、④川芎嗪1×10-2mg/ml组、⑤川芎嗪1×10-1mg/ml组、⑥川芎嗪1mg/ml组，在37℃、5%CO2培养箱中继续培养24h、48h、72h，用天狼猩红染色，酶标仪检测细胞在540nm波长的吸光度A值，通过比较A值观察MRC-5细胞内胶原蛋白表达的变化。通过体外培养MRC-5细胞，分为①正常对照组、②地塞米松组（5×10-2mg/ml）③川芎嗪低浓度组（1×10-2mg/ml）、④川芎嗪高浓度组（1mg/ml），通过细胞爬片免疫组化法观察细胞内Ⅰ型胶原蛋白（COLⅠ）、Ⅲ型胶原蛋白（COLⅢ）表达的变化，并采用Image pro-plus图像分析系统计算出各组COLⅠ、COLⅢ表达的IOD。通过体外培养MRC-5细胞，分为①正常对照组、②地塞米松组（5×10-2mg/ml）③川芎嗪低浓度组（1×10-2mg/ml）、④川芎嗪高浓度组（1mg/ml），用实时定量PCR法测定各组MRC-5细胞内COLⅠ m RNA、COL Ⅲ mRNA表达的变化。结果：1.盐酸川芎嗪各浓度组：1×10-3mg/ml组、1×10-2mg/ml组、1×10-1mg/ml组、1mg/ml组，分别与MRC-5细胞共孵育24h、48h、72h后，发现随着川芎嗪浓度的增大或川芎嗪与MRC-5细胞共孵育时间的延长，细胞增殖抑制率改变明显。同一时间下1mg/ml组细胞增殖抑制率最高，同一浓度川芎嗪作用下72h后细胞增殖抑制率最高；而且盐酸川芎嗪1mg/ml组细胞增殖抑制率随着时间的延长，逐渐升高。2.天狼猩红法染色，酶标仪检测540nm处的吸光度A值，观察各组MRC-5细胞内胶原蛋白表达变化情况，发现共孵育24h后正常对照组细胞胶原蛋白合成分泌较少，与之相比较各药物组细胞内胶原蛋白表达未见明显变化。发现共孵育48h后正常对照组细胞内胶原蛋白合成分泌的明显增多，与之相比较各药物组细胞内胶原蛋白的表达明显减少；其中川芎嗪1mg/ml浓度组比其它几组抑制作用都强，与地塞米松组抑制作用相当。发现共孵育72h后正常对照组细胞内胶原蛋白合成分泌有所下降，与正常对照组相比较川芎嗪1mg/ml浓度组胶原蛋白的表达明显减少，并与地塞米松组表达相当。3.免疫组化法检测MRC-5细胞内COLⅠ蛋白、COLⅢ蛋白表达变化情况，采用Image pro-plus图像分析系统计算出各组COLⅠ蛋白、COLⅢ蛋白表达的IOD，通过统计学分析发现地塞米松组、川芎嗪低浓度组（1×10-2mg/ml）、川芎嗪高浓度组（1mg/ml）各组MRC-5细胞COLⅠ蛋白、COLⅢ蛋白表达的IOD与正常对照组相应的IOD相比较均有所减少，且差别有统计学意义（P<0.05）；川芎嗪高浓度组（1mg/ml）与地塞米松组比较差别无统计学意义。4.实时定量PCR检测MRC-5细胞内COL Ⅰm RNA、COLⅢ m RNA相对表达量，结果发现地塞米松组、川芎嗪高浓度组、川芎嗪低浓度组各组MRC-5细胞内COL Ⅰm RNA、COL Ⅲm RNA相对表达量与正常对照组的相对表达量比较均有所减少，且差别有统计学意义（P<0.05），川芎嗪高浓度组同川芎嗪低浓度组、地塞米松组分别比较差别有统计学意义（P<0.05）。结论：1．盐酸川芎嗪注射液可以抑制人胚肺成纤维细胞系MRC-5细胞的增殖，减少其沉积肺内，起到防治肺纤维化的作用。2．盐酸川芎嗪注射液具有抗肺纤维化的作用，其机制可能是通过抑制MRC-5细胞COL Ⅰm RNA、COLⅢ m RNA的表达，进而抑制细胞内胶原蛋白的合成分泌，减少胶原蛋白肺内沉积，来防治肺纤维化。更多还原

【Abstract】 ObjectiveThe research aimed to investigate effects of different concentrations of TMP on MRC-5cells proliferation through in vitro cell culture, and then observed the changes of expression ofcollagen protein in MRC-5after different concentrations of TMP for different time. Theresults will provide new theory and ideas for traditional Chinese medicine used in preventionand treatment of pulmonary fibrosis.Methods1. Human embryonic lung fibroblast cell line (MRC-5) were cultured and then weretreated with different concentrations of ligustrazine. The cells were divided into five groups:①normal control group,②l igustrazine1×10-3mg/mL group;③l igustrazine1×10-2mg/mLgroup;④ligustrazine1×10-1mg/mL group;⑤ligustrazine1mg/mL group. After cultured24h,48h and72h, the morphological changes of cells were observed under microscope, and thenthe MTT method was used to detect the proliferation of MRC-5after treatment of differentconcentrations of ligustrazine for different time.2. MRC-5cells were divided into:①normal control group,②the dexamethasone group,③ligustrazine1x10-3mg/mL group,④ligustrazine1x10-2mg/mL group,⑤ligustrazine1x10-1mg/mL group,⑥ligustrazine1mg/mL group. After developed in37℃5%CO2incubator for24h,48h and72h, cells were stained with Sirius scarlet and the collagen proteinin cells was observed. Then intracellular protein expression of COLⅠ a nd COL Ⅲwasanalyzed using immunohistochemical method and q RT-PCR was used to detect the mRNAexpression of COL Ⅰ and COL Ⅲ.Results1. After MRC-5cells were treated with different concentrations of ligustrazine for24h,48h and72h, it was found that with the increasing of concentration of the drug and theprolong of the incubation time, rate of cells inhibition increased significantly. Furthermore,under the same treatment interval, rate of cell proliferation inhibition of1mg/mL group ishighest, and under the same concentration, rate of cell proliferation inhibition was mostsignificant after72h treatment. The rate of cell proliferation inhibition of cells increasedgradually over time.2. Sirius scarlet method revealed that with the increase of concentration and the extension of interaction time of ligustrazine, the collagen synthesis was not decreasedsignificantly after treated for24h, while after treated for48h, significant decrease of collagensynthesis was observed, and ligustrazine1mg/mL group exerted most inhibition effects thanother groups as well as dexamethasone group. After incubated for72h, ligustrazine1mg/mLgroup still exhibit strong inhibition effect to collagen synthesis as well as dexamethasonegroup.3. Immunohistochemical method revealed that the expression of COLⅠ protein andCOL Ⅲ protein were suppressed significantly in dexamethasone group, ligustrazine highconcentration group (1mg/ml) and ligustrazine low concentration group (1x10-2mg/ml)compared with normal control group. But the difference in COLⅠ protein and COLⅢprotein expression between dexamethasone group and ligustrazine high concentration group(1mg/ml) was not statistically significant.4. qRT-PCR found that mRNA expression of COLⅠ and COL Ⅲwas significantlyinhibited in dexamethasone group, ligustrazine high concentration group (1mg/ml) andligustrazine low concentration group (1x10-2mg/ml) compared with normal control group,and mRNA expression in ligustrazine high concentration group (1mg/ml) decreased obviouslycompared with ligustrazine low concentration group (1x10-2mg/ml) and dexamethasonegroup.Conclusion:1. Ligustrazine Hydrochloride Injection can inhibit human embryonic lung fibroblast cellline (MRC-5) proliferation and reduce the lung deposition of the cells, which may contributesto the prevention and treatment of pulmonary fibrosis.2. Ligustrazine Hydrochloride Injection has Anti-pulmonary Fibrosis Effect. Themechanism may be associated with inhibiting of the expression of COL ⅠmRNA andCOL ⅢmRNA and protein, and then inhibits the synthesis of intracellular collagen secretion,reduce collagen deposition to the lungs.更多还原