摘要
[目的]：
观察从中药三七中获得的代号为NV-KS (Ginsenoside C-K)的提取物对云南宣威肺腺癌细胞株XWLC-05(Xuanwei Lung Cancer-05)的体外抑制作用,并探讨NV-KS对XWLC-05细胞的作用机制。
[方法]：
1.采用四甲基偶氮唑蓝(MTT)法测定不同时间和不同浓度NV-KS和CDDP对XWLC-05细胞、GLC细胞及KMB-17细胞生长的抑制作用,并将每种细胞分为四组即阴性对照组、溶剂对照组、实验组(加入NV-KS)和阳性对照组(加入CDDP),确定半数抑制浓度(IC50)；
2. XWLC-05细胞用NV-KS及CDDP诱导72h后,凯基活细胞/凋亡细胞/坏死细胞鉴别试剂盒(AO/EB法.荧光显微镜)观察细胞凋亡状况；
3. XWLC-05细胞经NV-KS及CDDP诱导72h,固定液处理,电镜扫描,观察细胞凋亡小体；
4. XWLC-05细胞及KMB-17细胞经NV-KS及CDDP诱导72h,用GENMED细胞凋亡DNA条带检测试剂盒处理,检测凋亡条带。
5.数据统计和分析采用SPSS17.0软件包进行。数据以均数±标准差(x±s)表示,组间资料应用单因素方差分析(ONE WAY ANOVA)和t检验。
[结果]：
1. NV-KS及CDDP对XWLC-05细胞、GLC细胞及KMB-17细胞的体外抑制作用呈剂量和时间依赖关系,数值如下表：
上述数据顺铂组两两比较p值无统计学意义,药物NV-KS组两两比较,XWLC-05与KMB-17比较p<0.05(p=0.012),有统计学意义。在后期的实验研究中,重点做NV-KS诱导XWLC-05细胞的凋亡实验；
2. NV-KS及CDDP诱导细胞72h后经AO/EB法处理后,荧光显微镜观察,均有凋亡细胞产生；
3. NV-KS及CDDP诱导细胞72h后固定液处理,电镜扫描图片显示有凋亡细胞；
4. NV-KS及CDDP诱导细胞72h后经GENMED细胞凋亡DNA条带检测试剂盒处理,可见DNA片断化改变的“梯形”条带。
[结论]：
1. NV-KS能抑制XWLC-05细胞体外增殖,其抑制作用呈剂量和时间依赖性；
2. NV-KS能促进XWLC-05细胞的凋亡,形成新月形的凋亡早期细胞,也可形成胞质芽状突起的凋亡晚期细胞；
3. NV-KS能促进XWLC-05细胞的凋亡,形成凋亡小体；
4. NV-KS能促进XWLC-05细胞的凋亡,形成明显的断裂条带,而在一定的浓度范围内作用于KMB-17细胞不能形成断裂条带。
[Objective]:
This study was designed to primarily investigate the inhibitory effects and the mechanisms of NV-KS abstracted from Panax Notoginseng of Yunnan on lung cancer cell line XWLC-05and to eluminate its potential mechanism in apoptosis.
[Methods]:
1. MTT (Methy thiazolyl tetrazolium) assay was used to evaluate the inhibitory effects of XWLC-05、GLC and KMB-17induced by NV-KS and CDDP in different time and different concentration, and each one is divided into four groups of negative control group、solvent control group、 and experimental group(with the NV-KS)、 positive control group(with the CDDP),and then to calculate the half inhibiting concentration(ICso).
2. XWLC-05were induced by NV-KS and CDDP in72hours,KeyGEN living cells/apoptotic cells/necrotic cells identification kit(AO/EB method)by fluorescence microscopy was used to observate apoptosis of the cells.
3. XWLC-05were induced by NV-KS and CDDP in72hours and then put in fixed liquid, electron microscope was used to scan the cells.
4. DNA ladder of XWLC-05and KMB-17cells were induced by NV-KS and CDDP in72hours was tested by GENMED DNA testing kit.
5. All data were analyzed by SPSS17.0software and statistics were expressed as mean±standard deviation (χ±s), single factor analysis of variance (ONE of WAY ANOVA) and t test were used to get the statistical significance.
[Results]:
1. NV-KS and CDDP could inhibit the proliferation of XWLC-05、 GLC and KMB-17cells, on dose-and time-dependenant manner in vitro, the values of IC50for each group are exhibited in following table:
There is no statistic significancance between cisplatin and pairwise group (p>0.05), but abvious difference of IC50was found in NV-KS cells betwen XWLC-05and KMB-17treatment (p=0.012; p<0.05).
2. The apoptosis XWLC-05cells induced by NV-KS and CDDP at72hours were found by fluorescence microscope with AO/EB method.
3. Apoptotic bodies of XWLC-05induced by NV-KS and CDDP at72hours were deteced by electron microscopy images.
4.. DNA ladders were found in XWLC-05and KMB-17cells treated with GENMED.
[Conclusion]:
1. NV-KS could inhibit the proliferation of the XWLC-05cells in vitro, its proliferation is on time and dose dependment.
2. NV-KS could inhibit the proliferation of the XWLC-05cells in vitro,apoptosis in the early formation of crescent-shaped cells, and also the formation of apoptosis in late cell cytoplasm bud-like protuberances.
3. NV-KS could inhibit the proliferation of the XWLC-05cells in vitro, apoptotic bodies of cells can form.
4. NV-KS can induce XWLC-05cells apoptosis and form fracture bands, but to KMB-17it can not form the fracture bands within a certain concentration.
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