吉林省双阳梅花鹿鹿茸蛋白组分的细胞毒性实验研究

目的探讨吉林双阳梅花鹿鹿茸蛋白组分对肿瘤细胞的细胞毒性作用。方法将新鲜鹿茸高速匀浆上清透析后,用超滤离心法获取分子量小于10kD,大于10kD小于30kD,大于30kD的3种蛋白组分;采用MTT法,检测3种蛋白组分对人肝癌SMMC-7721细胞系及人肺腺癌SPC-A-1细胞系的细胞毒性作用。另外,设鹿茸提取物原浓度的1/2,1/4,1/8为高,中,低3个浓度组,以0.01ml/g浓度皮下注射昆明小鼠,连续注射14天后,取小鼠脾细胞培养(培养基含ConA0.005mg/ml)72h后,加入MTT(5mg/ml)0.01ml,继续培养4h,测定OD值,计算淋巴细胞转化值。结果针对SMMC-7221细胞,分子量<10kD,>30kD,10kD<<30kD的鹿茸蛋白组分中,高剂量组的抑制率均大于30%,分子量<10kD,10kD<<30kD的鹿茸蛋白组分中,中高剂量组的抑制率大于30%,而且呈现剂量依赖性关系。针对SPC-A-1细胞,从所选取的高剂量至低剂量的抑制率均小于30%。鹿茸蛋白组分高、中、低剂量组的平均OD值分别为0.46±0.05、0.29±0.03和0.17±0.03;胸腺肽阳性对照组的平均OD值为0.47±0.07;生理盐水阴性对照组的平均OD值为0.12±0.04。鹿茸蛋白组分高、中、低剂量组的转化值分别为0.34、0.17和0.05;胸腺肽阳性对照组的转化值为0.35。鹿茸蛋白组分的高剂量组和胸腺肽对照组OD值比较,差异不具有显著性(P>0.05)。结论对于SPC-A-1细胞,鹿茸各蛋白组分无明显细胞毒性作用,而对于SMMC-7221细胞,各蛋白组分的高剂量、中高剂量组则呈现一定的细胞毒性作用。鹿茸蛋白组分高剂量组具有与胸腺肽注射液相近的促进淋巴细胞转化活性。

Cytotoxicity studies of protein components from Jilin Shuangyang sika deer pilose antler

Objective To study the cancer cell cytotoxicity mediated by protein components from Jilin Shuangyang sika deer pilose antler.Methods The raw tissue of pilose antler was homogenated with high speed.The supernatant was obtained by centrifuge.After dialyzing,ultrafiltration was applied to process the total protein solution.Then,various components with various molecular weight（less than 10 kD,high than 10 kD and less than 30 kD,higher than 30 kD） were obtained.Cytotoxicity of human lung adenocarcinoma SPC-A-1 cells and human liver cancer cell SMMC7721 mediated by protein components was to be studied.There are three experimential groups include high,middle and low dose groups（1/2,1/4 and 1/8 original deer pilose antler extract,respectively）.After 14 days subcutaneous injection（0.01 ml/g） in KunMing rats,spleen cells are cultured for 72 hours（The medium contains ConA 0.005 mg/ml）.Then 0.01ml MTT（5 mg/ml） is added into medium.After 4 hour culture,OD value and lymphocyte transformation value are assayed.Results As for SMMC-7221 cell line,inhibitory rates of all high dose groups in every experimental groups（less than 10 kD,higher than 10 kD and less than 30 kD,higher than 30 kD） are greater than 30%.Inhibitory rates of sub high dose groups in two experimental groups（less than 10 kD,higher than 10 kD and less than 30 kD） are greater than 30% and follows relationship of dose-dependence.As for SPC-A-1 cell line,inhibitory rates of all of the groups are less than 30%.OD value of high,middle and low dose groups are 0.46±0.05、0.29±0.03 and 0.17±0.03,respectively.OD value of thymosin treatment group is 0.47±0.07.OD value of physiological saline treatment group is 0.12±0.04.Lymphocyte transformation value of high,middle and low dose groups are 0.34、0.17 and 0.05.There is not obvious difference between high dose group and thymosin treatment group（P〈0.05）.Conclusion Taken together,there is no significant cytotoxicity on SPC-A-1 mediated by every protein components.While there is definite cytotoxicity on SMMC-7721 mediated by protein components within high dose and sub high dose groups.High dose group of pilose antler protein may improve the activity of lymphocyte transformation similar to thymosin.