羟基红花黄色素A对体外培养子宫内膜异位症在位内膜细胞增殖及细胞因子表达的影响
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摘要
目的:以不同浓度羟基红花黄色素A(HSYA)干预体外培养子宫内膜异位症(Endometriosis, EMs)在位子宫内膜细胞。四甲基偶氮唑蓝法(MTT)检测HSYA对在位子宫内膜细胞的生长增殖的影响,酶联免疫吸附法(ELISA)测定HSYA对细胞培养上清液中细胞因子VEGF、bFGF分泌的影响。从而探讨HSYA治疗子宫内膜异位症的可行性。
方法:
1研究对象
1.1实验组:选2009年10月—2010年1月在我院行腹腔镜或开腹手术治疗的EMs患者(术后病理证实均为增殖期),共15例(38.13±7.26岁)。
1.2对照组:选择同期在我院因、纵隔子宫、畸胎瘤、单纯囊肿经腹腔镜检查无盆腔子宫内膜异位症(术后病理证实正常子宫内膜增殖期)者,共14例(34.92±8.11岁)。
两组患者月经周期正常,近6个月无服用激素类药物及免疫抑制类药物史及IUD置入史。排除:子宫内膜息肉、急慢性盆腔炎、乳腺癌、子宫内膜癌等其它妇科恶性肿瘤;内分泌、免疫及代谢性疾病;高血压、心脏病、肾病等内科疾病等。采取标本均得到患者的同意,并签署了知情同意书。
2标本采集
刮取子宫内膜,立即置于冰浴的含青霉素和链霉素的1:1DMEM/F12培养基中,2小时内送实验室进行培养。
3实验方法
3.1细胞培养
子宫内膜组织经漂洗、分离、消化、过滤。台盼兰染色,鉴定细胞活力。接种于含10%NBCS、青霉素及链霉素的FD的细胞培养瓶中,孵育24h换液,弃去未贴壁细胞及红细胞,继续培养,每隔48h换液一次,倒置显微镜下观察细胞形态变化及生长状况。
细胞长满80%瓶壁时,胰蛋白酶消化后,传代培养。细胞波形蛋白抗体及角蛋白抗体进行免疫化学细胞鉴定。
3.2细胞生长测定
3.2.1正常生长曲线
取第2-3代细胞接种于96孔培养板,并设凋零孔。培养24、48、72、96、120、144小时后,每孔加入MTT、二甲基亚砜(DMSO),酶联免疫检测仪测定细胞吸光度值(OD值),绘制细胞生长曲线。
3.2.2药物干预后细胞生长曲线
取第2-3代细胞接种于96孔培养板,24h贴壁后加入不同浓度的GnRHa和HSYA ,每一浓度设5个复孔,并设空白组。培养24、48、72h后加MTT、DMSO,测定OD值,绘制细胞生长曲线。
3.3 ELISA法测定细胞因子
取第2-3代细胞接种于24孔培养板,含10%NBCS的FD培养24h,换含2%NBCS的FD继续培养24h,弃上清液,分别加入含不同浓度的GnRHa和HSYA的FD培养基,设立空白对照组,每组设3个复孔,48h后收集细胞培养上清液,于-20℃冻存待测, ELISA法测定VEGF及bFGF的含量。
结果:
1细胞生长情况
EMs在位内膜细胞培养成功率为93.3%,正常子宫内膜细胞培养成功率为92.9%,细胞活力均在90%以上。
2细胞鉴定
①基质细胞Vimentin染色阳性,细胞胞浆呈棕黄色。②腺上皮细胞Cytokeratin染色阳性,细胞胞浆呈棕黄色。③阴性对照:PBS作为一抗,Vimentin及Cytokeratin染色均阴性。
倒置显微镜下观察,子宫内膜细胞接种0.5h后即开始贴壁,24h后基本贴壁完全并开始生长。约2-4天细胞生长加快,以后生长减慢开始进入平台期。细胞紧密连接,排列较规则。
3药物对离体细胞生长的影响
3.1 HSYA对离体细胞生长的抑制作用
药物作用24h后,细胞生长受到抑制,但无显著差异。
药物作用48h、72h后,HSYA显著抑制细胞生长,呈剂量依赖性即HSYA浓度越大OD值越小(与细胞数成正比)。
随着HSYA作用时间的延长及药物浓度的增加,对细胞的抑制作用逐渐增强。
3.2 GnRHa对离体细胞生长的抑制作用
药物作用24h后,细胞生长受到抑制,但无显著差异。
药物作用48h、72h后,细胞生长受到显著抑制,呈剂量依赖性。随着GnRHa作用时间的延长及药物浓度的增加,对细胞的抑制作用逐渐增强。
3.3 HSYA和GnRHa对离体细胞生长的抑制作用的比较
药物作用24h后,两种药物对细胞有抑制作用但无明显差异。
48h后HSYA 20、2、0.2μg/ml组分别与GnRHa10、1、0.1μg/ml组同级比较,GnRHa对细胞的抑制作用强于HSYA。HSYA 20μg/ml组比GnRHa 1μg/ml组抑制作用强,HSYA 2μg/ml组比GnRHa 0.1μg/ml组对细胞的抑制作用强。
72h后HSYA 20、2、0.2μg/ml组分别与GnRHa10、1、0.1μg/ml组同级比较,GnRHa对细胞的抑制作用强于HSYA。HSYA 20μg/ml组与GnRHa 0.1μg/ml组抑制作用强。HSYA 2μg/ml组比GnRHa 0.01μg/ml组对细胞的抑制作用有统计学意义。
4两种药物作用于正常子宫内膜细胞虽然有一定的抑制作用,但并没有统计学意义。
5药物对细胞培养上清液中细胞因子表达的影响
5.1 HSYA和GnRHa对细胞培养上清液中VEGF表达的抑制作用与不加药对照组比较,HSYA和GnRHa各浓度组均显著抑制VEGF的表达,呈剂量依赖性。
两种药物抑制作用的比较,HSYA 20、0.2μg/ml组分别比GnRHa 0.1、0.01μg/ml组抑制作用强。因此虽然GnRHa对VEGF的抑制作用比HSYA强,但在一定范围内HSYA比GnRHa作用强。
5.2 HSYA和GnRHa对细胞培养上清液中bFGF表达的抑制作用与不加药组比较,HSYA20、2μg/ml组和GnRHa10、1μg/ml组均显著抑制bFGF的表达,呈剂量依赖性。
两种药物抑制作用的比较,HSYA 20、2μg/ml组对bFGF表达的抑制作用比GnRHa组10、1μg/ml组对bFGF强有统计学意义。由此可见,HSYA对bFGF的抑制作用比GnRHa强。
结论:
1子宫内膜细胞体外培养模型成功建立。
2子宫内膜细胞分为间质细胞及腺上皮细胞两种。基质细胞中波形蛋白染色阳性,腺上皮细胞中角蛋白染色阳性。细胞悬液中基质细胞为单个细胞,圆形。腺上皮细胞为细胞团,类似葡萄串状。
3细胞接种后约0.5小时开始贴壁,大约24小时细胞贴壁完成。2-4天细胞生长加快,第5天以后细胞生长进入平台期。
4 HSYA及GnRHa均能显著抑制体外培养的EMs在位子宫内膜细胞的增殖,呈剂量-时间依赖性。同级比较GnRHa比HSYA抑制作用强,但在一定的浓度范围内HSYA比GnRHa对细胞的抑制作用强。
5 HSYA及GnRHa对EMs在位内膜细胞体外培养上清液中细胞因子VEGF及bFGF的表达均具有抑制作用,呈剂量依赖性。GnRHa对VEGF的抑制作用强于HSYA,HSYA对bFGF的抑制作用强于GnRHa。
6两种药物作用于正常子宫内膜细胞虽然有一定的抑制作用,但并没有统计学意义。
HSYA对EMs离体在位子宫内膜细胞增殖的抑制作用可能是通过其对VEGF及bFGF表达的下调实现的。提示HSYA可能是治疗EMs的潜在药物,为子宫内膜异位症的治疗提供新的理论基础。
Objective: Different concentrations of HSYA interfered in vitro endometriosis (Endometriosis, EMs) eutopic endometrial cells, the growth and proliferation of eutopic endometrial cells were measured by Blue tetrazolium method (MTT) test.
The expression of the VEGF protein and the bFGF protein in the medium induced by HSYA were analyzed by enzymelinked immunosorbent assay (ELISA).To explore the feasibility of HSYA for the treatment of endometriosis.
Methods:
1 The study subject:
1.1 The study group: patients who undergoing laparotomy or laparoscopy for endometriosis in our hospital, selected from October 2009 to February 2010(proliferative phase were pathologically confirmed), a total of 15 cases (mean age [±SD] 38.13±7.26years old).
1.2 The control group: Selected the patients in the same period in our hospital with septate uterus, ovarian dysembryoma or simple cyst and without pelvic endometriosis (normal proliferative phase endometrium were pathologically confirmed), a total of 14 cases (mean age [±SD] 34.92+8.11years old).
All of the women in the two groups had normal menstrual cycles, nearly 6 months without taking steroids and immunosuppressive drugs history and the history of IUD placement. The patients with endometrial polyps, acute or chronic pelvic inflammatory diseases, breast cancer, endometrial cancer and other gynecological malignancies were excluded. All of them had not endocrine, immune and metabolic diseases and high blood pressure, heart disease, kidney disease etc. Samples were taken by the patient's consent, and signed informed consent.
2 Specimen collecting:
The endometrium samples were scraped and placed in ice- bath 1:1 DMEM/F12 medium containing penicillin and streptomycin, sent to the laboratory to culture in 2 hours.
3 Experiment methods:
3.1 Cell culture
Endometrial tissue was rinsed, separated, digested, filtrated.The cells were cultured in flasks, which contained DMEM/F-12 medium supplemented with 10% NBCS, penicillin and streptomycin at 37°C in humidified atmosphere of 5% CO2 in air. After 24 hours replaced the medium, discarded the no adherent cells and red blood cells, then changed the medium every other day. Under the Inverted microscope observed cell morphology and growth conditions.
When the cells covered 80% of the flasks wall, were digested by trypsin, and plated in flasks in the same agent to culture, changed the medium every other day. The ECs were identified byimmunohistochemical staining, with markers specific to ESCs (Vimentin), epithelial cells (Cytokeratin).
3.2 Cell proliferation assay:
3.2.1 The normal growth curve:
Took the first 2-3 generations ECs to seed in 96-microwell plates, and set a withered well. And incubated for 24h,48h,72h, 96h,120h,144h, added MTT and Dimethyl Sulfoxide per well. Enzyme-linked immunosorbent detector determined the cell absorbance (OD value) at 492nm wavelength. Draw cell growth curve by Microsoft Excel software.
3.2.2 The cell growth curve after the intervention of the medicines
Took the first 2-3 generations ECs to seed in 24-microwell plates, after 24h incubation, the cells were washed by PBS. Then added HSYA and GnRHa, cultured for 24h, 48h, 72h. Each group set up five wells, and a control group. Cultured for an additional 24h, 48h, 72h. The absorbance values were determined at 492 nm wavelength. Microsoft Excel software draw cell growth curve.
3.3 Measurement of VEGF and bFGF by ELISA
Took the first 2-3 generations ECs to seed in 24-microwell plates, and set a control well and incubated with 10% NBCS FD medium for 24 h, subsequently washed and incubated with medium supplemented with 2% NBCS for an additional 24 h under basal conditions, after the addition of serum-free FD with different concentrations of GnRHa and HSYA for 48 h. Each group set up three wells. The control group did not add any drugs. Culture supernatants were collected and kept at -20°C to be measured. The VEGF and bFGF concentration in culture supernatants was quantitated using a commercial ELISA kit specific for human VEGF/bFGF.
Results:
1 Cell growth condition:
Endometrial cells from EMs patients were successfully cultured, and the achievement ratio was 93.3% (14/15). The achievement ratio of the normal endometrial cells was 92.9% (13/14). The viability was at least 90%.
2 Identification of cells in immunocytochemistry
①Endometrial stromal cells stainned positive for vimentin, cell cytoplasm showed brown.②Endometrial glandular epithelial cells stainned positive for Cytokeratin, cell cytoplasm showed brown.③Negative control:
PBS as antibody,Vimentin and Cytokeratin stainned negative。Inverted microscope observed the cells. Endometrial cells began to adhere after 0.5h and 24h later basically completed adherent and began to grow. About 2-4days cells grew to speed up, entered the platform period, and the cells linked tightly with each other.
3 The effect of HSYA and GnRHa on the proliferation of eutopic endometrial cells
3.1 The effect of HSYA on the proliferation of in vitro cells
After 24h, HSYA could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.
After 48h, 72h, HSYA significantly inhibited cell growth in a dose dependent manner, the concentration of HSYA were higher the smaller of the OD value (proportional to cell number).
HSYA significantly inhibited the proliferation of the ECs with the concentration and time increased.
3.2 The effect of GnRHa on the proliferation of in vitro cells After 24h, GnRHa could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.
After 48h, 72h, GnRHa significantly inhibited cell growth in a dose dependent manner. GnRHa significantly inhibited the proliferation of the ECs with concentration and time increased.
3.3 The effect of HSYA and GnRHa on the proliferation of in vitro cells
After 24h, both drugs inhibited the proliferation of the ECs but no significant difference.
After 48h, the HSYA20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group.
After 72h, the HSYA 20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 0.01μg/ml group.
4 In spite of both drugs inhibited normal endometrial cells, had no significant distinction.
5. The expression of cytokine in cell culture supernatant
5.1 The expression of VEGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.
Compared With the control group without drugs, various concentrations groups all significantly inhibited the expression of VEGF in a dose dependence manner.
Comparison of the inhibition of the two drugs, HSYA 20, 0.2μg/ml groups had stronger inhibition than GnRHa 0.1,0.01μg/ml groups.Thus, although the inhibition of GnRHa on the expression of VEGF stronger than HSYA, but in a certain concentration range HSYA was stronger than the GnRHa.
5.2 The expression of bFGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.
Compared With the control group without drugs, HSYA 20,2μg/ml groups and GnRHa10, 1μg/ml groups significantly inhibited the expression of bFGF in a dose dependence manner.
Comparison of the inhibition of the two drugs, HSYA 20、2μg/ml groups inhibited the expression of bFGF significantly stronger than the GnRHa 10、1μg/ml groups. Thus HSYA had stronger inhibition on the expression of bFGF than GnRHa.
Conclusion:
1 An in vitro model of endometrial cells successfully established.
2 Endometrial cells included stromal cells and glandular epithelial cells.
Stromal cells stained positive for vimentin, epithelial cells stained positive for cytokeratin. In the cell suspension, the stromal cells were single, round cells. The gland epithelial cells were multiple cell clusters, thyrsiform.
3 Endometrial cells began adherent after 0.5h and about 24h later basically completed adherent and began to grow. About 2-4days cells grew speed up, after 5-6 days entered the platform period, and the cells linked tightly with each other.
4 HSYA and GnRHa both significantly inhibited the proliferation of the ECs in a dose-time dependence manner. Comparison of HSYA at the same level, GnRHa had stronger inhibition, but in a certain concentration range HSYA inhibited the proliferation of the ECs than GnRHa stronger.
5 Both HSYA and GnRHa inhibited the expression of VEGF and bFGF in cell culture supernatant dose-dependent. GnRHa inhibited the expresstion of VEGF stronger than HSYA. HSYA inhibited the expresstion of bFGF stronger than GnRHa.
6 Both drugs could inhibited normal endometrial cells, had no statistically significant.
HSYA inhibited the proliferation of endometrial cells from EMs patients may be through its down regulation the expression of VEGF and bFGF implementation. Prompted HSYA may be a potential drug for the treatment of endometriosis, to provide a new theoretical basis for the theropy of endometriosis.
方法:
1研究对象
1.1实验组:选2009年10月—2010年1月在我院行腹腔镜或开腹手术治疗的EMs患者(术后病理证实均为增殖期),共15例(38.13±7.26岁)。
1.2对照组:选择同期在我院因、纵隔子宫、畸胎瘤、单纯囊肿经腹腔镜检查无盆腔子宫内膜异位症(术后病理证实正常子宫内膜增殖期)者,共14例(34.92±8.11岁)。
两组患者月经周期正常,近6个月无服用激素类药物及免疫抑制类药物史及IUD置入史。排除:子宫内膜息肉、急慢性盆腔炎、乳腺癌、子宫内膜癌等其它妇科恶性肿瘤;内分泌、免疫及代谢性疾病;高血压、心脏病、肾病等内科疾病等。采取标本均得到患者的同意,并签署了知情同意书。
2标本采集
刮取子宫内膜,立即置于冰浴的含青霉素和链霉素的1:1DMEM/F12培养基中,2小时内送实验室进行培养。
3实验方法
3.1细胞培养
子宫内膜组织经漂洗、分离、消化、过滤。台盼兰染色,鉴定细胞活力。接种于含10%NBCS、青霉素及链霉素的FD的细胞培养瓶中,孵育24h换液,弃去未贴壁细胞及红细胞,继续培养,每隔48h换液一次,倒置显微镜下观察细胞形态变化及生长状况。
细胞长满80%瓶壁时,胰蛋白酶消化后,传代培养。细胞波形蛋白抗体及角蛋白抗体进行免疫化学细胞鉴定。
3.2细胞生长测定
3.2.1正常生长曲线
取第2-3代细胞接种于96孔培养板,并设凋零孔。培养24、48、72、96、120、144小时后,每孔加入MTT、二甲基亚砜(DMSO),酶联免疫检测仪测定细胞吸光度值(OD值),绘制细胞生长曲线。
3.2.2药物干预后细胞生长曲线
取第2-3代细胞接种于96孔培养板,24h贴壁后加入不同浓度的GnRHa和HSYA ,每一浓度设5个复孔,并设空白组。培养24、48、72h后加MTT、DMSO,测定OD值,绘制细胞生长曲线。
3.3 ELISA法测定细胞因子
取第2-3代细胞接种于24孔培养板,含10%NBCS的FD培养24h,换含2%NBCS的FD继续培养24h,弃上清液,分别加入含不同浓度的GnRHa和HSYA的FD培养基,设立空白对照组,每组设3个复孔,48h后收集细胞培养上清液,于-20℃冻存待测, ELISA法测定VEGF及bFGF的含量。
结果:
1细胞生长情况
EMs在位内膜细胞培养成功率为93.3%,正常子宫内膜细胞培养成功率为92.9%,细胞活力均在90%以上。
2细胞鉴定
①基质细胞Vimentin染色阳性,细胞胞浆呈棕黄色。②腺上皮细胞Cytokeratin染色阳性,细胞胞浆呈棕黄色。③阴性对照:PBS作为一抗,Vimentin及Cytokeratin染色均阴性。
倒置显微镜下观察,子宫内膜细胞接种0.5h后即开始贴壁,24h后基本贴壁完全并开始生长。约2-4天细胞生长加快,以后生长减慢开始进入平台期。细胞紧密连接,排列较规则。
3药物对离体细胞生长的影响
3.1 HSYA对离体细胞生长的抑制作用
药物作用24h后,细胞生长受到抑制,但无显著差异。
药物作用48h、72h后,HSYA显著抑制细胞生长,呈剂量依赖性即HSYA浓度越大OD值越小(与细胞数成正比)。
随着HSYA作用时间的延长及药物浓度的增加,对细胞的抑制作用逐渐增强。
3.2 GnRHa对离体细胞生长的抑制作用
药物作用24h后,细胞生长受到抑制,但无显著差异。
药物作用48h、72h后,细胞生长受到显著抑制,呈剂量依赖性。随着GnRHa作用时间的延长及药物浓度的增加,对细胞的抑制作用逐渐增强。
3.3 HSYA和GnRHa对离体细胞生长的抑制作用的比较
药物作用24h后,两种药物对细胞有抑制作用但无明显差异。
48h后HSYA 20、2、0.2μg/ml组分别与GnRHa10、1、0.1μg/ml组同级比较,GnRHa对细胞的抑制作用强于HSYA。HSYA 20μg/ml组比GnRHa 1μg/ml组抑制作用强,HSYA 2μg/ml组比GnRHa 0.1μg/ml组对细胞的抑制作用强。
72h后HSYA 20、2、0.2μg/ml组分别与GnRHa10、1、0.1μg/ml组同级比较,GnRHa对细胞的抑制作用强于HSYA。HSYA 20μg/ml组与GnRHa 0.1μg/ml组抑制作用强。HSYA 2μg/ml组比GnRHa 0.01μg/ml组对细胞的抑制作用有统计学意义。
4两种药物作用于正常子宫内膜细胞虽然有一定的抑制作用,但并没有统计学意义。
5药物对细胞培养上清液中细胞因子表达的影响
5.1 HSYA和GnRHa对细胞培养上清液中VEGF表达的抑制作用与不加药对照组比较,HSYA和GnRHa各浓度组均显著抑制VEGF的表达,呈剂量依赖性。
两种药物抑制作用的比较,HSYA 20、0.2μg/ml组分别比GnRHa 0.1、0.01μg/ml组抑制作用强。因此虽然GnRHa对VEGF的抑制作用比HSYA强,但在一定范围内HSYA比GnRHa作用强。
5.2 HSYA和GnRHa对细胞培养上清液中bFGF表达的抑制作用与不加药组比较,HSYA20、2μg/ml组和GnRHa10、1μg/ml组均显著抑制bFGF的表达,呈剂量依赖性。
两种药物抑制作用的比较,HSYA 20、2μg/ml组对bFGF表达的抑制作用比GnRHa组10、1μg/ml组对bFGF强有统计学意义。由此可见,HSYA对bFGF的抑制作用比GnRHa强。
结论:
1子宫内膜细胞体外培养模型成功建立。
2子宫内膜细胞分为间质细胞及腺上皮细胞两种。基质细胞中波形蛋白染色阳性,腺上皮细胞中角蛋白染色阳性。细胞悬液中基质细胞为单个细胞,圆形。腺上皮细胞为细胞团,类似葡萄串状。
3细胞接种后约0.5小时开始贴壁,大约24小时细胞贴壁完成。2-4天细胞生长加快,第5天以后细胞生长进入平台期。
4 HSYA及GnRHa均能显著抑制体外培养的EMs在位子宫内膜细胞的增殖,呈剂量-时间依赖性。同级比较GnRHa比HSYA抑制作用强,但在一定的浓度范围内HSYA比GnRHa对细胞的抑制作用强。
5 HSYA及GnRHa对EMs在位内膜细胞体外培养上清液中细胞因子VEGF及bFGF的表达均具有抑制作用,呈剂量依赖性。GnRHa对VEGF的抑制作用强于HSYA,HSYA对bFGF的抑制作用强于GnRHa。
6两种药物作用于正常子宫内膜细胞虽然有一定的抑制作用,但并没有统计学意义。
HSYA对EMs离体在位子宫内膜细胞增殖的抑制作用可能是通过其对VEGF及bFGF表达的下调实现的。提示HSYA可能是治疗EMs的潜在药物,为子宫内膜异位症的治疗提供新的理论基础。
Objective: Different concentrations of HSYA interfered in vitro endometriosis (Endometriosis, EMs) eutopic endometrial cells, the growth and proliferation of eutopic endometrial cells were measured by Blue tetrazolium method (MTT) test.
The expression of the VEGF protein and the bFGF protein in the medium induced by HSYA were analyzed by enzymelinked immunosorbent assay (ELISA).To explore the feasibility of HSYA for the treatment of endometriosis.
Methods:
1 The study subject:
1.1 The study group: patients who undergoing laparotomy or laparoscopy for endometriosis in our hospital, selected from October 2009 to February 2010(proliferative phase were pathologically confirmed), a total of 15 cases (mean age [±SD] 38.13±7.26years old).
1.2 The control group: Selected the patients in the same period in our hospital with septate uterus, ovarian dysembryoma or simple cyst and without pelvic endometriosis (normal proliferative phase endometrium were pathologically confirmed), a total of 14 cases (mean age [±SD] 34.92+8.11years old).
All of the women in the two groups had normal menstrual cycles, nearly 6 months without taking steroids and immunosuppressive drugs history and the history of IUD placement. The patients with endometrial polyps, acute or chronic pelvic inflammatory diseases, breast cancer, endometrial cancer and other gynecological malignancies were excluded. All of them had not endocrine, immune and metabolic diseases and high blood pressure, heart disease, kidney disease etc. Samples were taken by the patient's consent, and signed informed consent.
2 Specimen collecting:
The endometrium samples were scraped and placed in ice- bath 1:1 DMEM/F12 medium containing penicillin and streptomycin, sent to the laboratory to culture in 2 hours.
3 Experiment methods:
3.1 Cell culture
Endometrial tissue was rinsed, separated, digested, filtrated.The cells were cultured in flasks, which contained DMEM/F-12 medium supplemented with 10% NBCS, penicillin and streptomycin at 37°C in humidified atmosphere of 5% CO2 in air. After 24 hours replaced the medium, discarded the no adherent cells and red blood cells, then changed the medium every other day. Under the Inverted microscope observed cell morphology and growth conditions.
When the cells covered 80% of the flasks wall, were digested by trypsin, and plated in flasks in the same agent to culture, changed the medium every other day. The ECs were identified byimmunohistochemical staining, with markers specific to ESCs (Vimentin), epithelial cells (Cytokeratin).
3.2 Cell proliferation assay:
3.2.1 The normal growth curve:
Took the first 2-3 generations ECs to seed in 96-microwell plates, and set a withered well. And incubated for 24h,48h,72h, 96h,120h,144h, added MTT and Dimethyl Sulfoxide per well. Enzyme-linked immunosorbent detector determined the cell absorbance (OD value) at 492nm wavelength. Draw cell growth curve by Microsoft Excel software.
3.2.2 The cell growth curve after the intervention of the medicines
Took the first 2-3 generations ECs to seed in 24-microwell plates, after 24h incubation, the cells were washed by PBS. Then added HSYA and GnRHa, cultured for 24h, 48h, 72h. Each group set up five wells, and a control group. Cultured for an additional 24h, 48h, 72h. The absorbance values were determined at 492 nm wavelength. Microsoft Excel software draw cell growth curve.
3.3 Measurement of VEGF and bFGF by ELISA
Took the first 2-3 generations ECs to seed in 24-microwell plates, and set a control well and incubated with 10% NBCS FD medium for 24 h, subsequently washed and incubated with medium supplemented with 2% NBCS for an additional 24 h under basal conditions, after the addition of serum-free FD with different concentrations of GnRHa and HSYA for 48 h. Each group set up three wells. The control group did not add any drugs. Culture supernatants were collected and kept at -20°C to be measured. The VEGF and bFGF concentration in culture supernatants was quantitated using a commercial ELISA kit specific for human VEGF/bFGF.
Results:
1 Cell growth condition:
Endometrial cells from EMs patients were successfully cultured, and the achievement ratio was 93.3% (14/15). The achievement ratio of the normal endometrial cells was 92.9% (13/14). The viability was at least 90%.
2 Identification of cells in immunocytochemistry
①Endometrial stromal cells stainned positive for vimentin, cell cytoplasm showed brown.②Endometrial glandular epithelial cells stainned positive for Cytokeratin, cell cytoplasm showed brown.③Negative control:
PBS as antibody,Vimentin and Cytokeratin stainned negative。Inverted microscope observed the cells. Endometrial cells began to adhere after 0.5h and 24h later basically completed adherent and began to grow. About 2-4days cells grew to speed up, entered the platform period, and the cells linked tightly with each other.
3 The effect of HSYA and GnRHa on the proliferation of eutopic endometrial cells
3.1 The effect of HSYA on the proliferation of in vitro cells
After 24h, HSYA could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.
After 48h, 72h, HSYA significantly inhibited cell growth in a dose dependent manner, the concentration of HSYA were higher the smaller of the OD value (proportional to cell number).
HSYA significantly inhibited the proliferation of the ECs with the concentration and time increased.
3.2 The effect of GnRHa on the proliferation of in vitro cells After 24h, GnRHa could inhibit the proliferation of eutopic endometrial cells from women with endometriosis, but no statistical significance.
After 48h, 72h, GnRHa significantly inhibited cell growth in a dose dependent manner. GnRHa significantly inhibited the proliferation of the ECs with concentration and time increased.
3.3 The effect of HSYA and GnRHa on the proliferation of in vitro cells
After 24h, both drugs inhibited the proliferation of the ECs but no significant difference.
After 48h, the HSYA20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group.
After 72h, the HSYA 20,2,0.2μg/ml and GnRHa10,1,0.1μg/ml group compared respectively, GnRHa had stronger inhibition on cells in vitro than HSYA. HSYA 20μg/ml group inhibited the proliferation of the ECs stronger than GnRHa 0.1μg/ml group. HSYA 2μg/ml group inhibited the proliferation of the ECs stronger than the GnRHa 0.01μg/ml group.
4 In spite of both drugs inhibited normal endometrial cells, had no significant distinction.
5. The expression of cytokine in cell culture supernatant
5.1 The expression of VEGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.
Compared With the control group without drugs, various concentrations groups all significantly inhibited the expression of VEGF in a dose dependence manner.
Comparison of the inhibition of the two drugs, HSYA 20, 0.2μg/ml groups had stronger inhibition than GnRHa 0.1,0.01μg/ml groups.Thus, although the inhibition of GnRHa on the expression of VEGF stronger than HSYA, but in a certain concentration range HSYA was stronger than the GnRHa.
5.2 The expression of bFGF in supernatant was significantly decreased after the treatment of HSYA and GnRHa.
Compared With the control group without drugs, HSYA 20,2μg/ml groups and GnRHa10, 1μg/ml groups significantly inhibited the expression of bFGF in a dose dependence manner.
Comparison of the inhibition of the two drugs, HSYA 20、2μg/ml groups inhibited the expression of bFGF significantly stronger than the GnRHa 10、1μg/ml groups. Thus HSYA had stronger inhibition on the expression of bFGF than GnRHa.
Conclusion:
1 An in vitro model of endometrial cells successfully established.
2 Endometrial cells included stromal cells and glandular epithelial cells.
Stromal cells stained positive for vimentin, epithelial cells stained positive for cytokeratin. In the cell suspension, the stromal cells were single, round cells. The gland epithelial cells were multiple cell clusters, thyrsiform.
3 Endometrial cells began adherent after 0.5h and about 24h later basically completed adherent and began to grow. About 2-4days cells grew speed up, after 5-6 days entered the platform period, and the cells linked tightly with each other.
4 HSYA and GnRHa both significantly inhibited the proliferation of the ECs in a dose-time dependence manner. Comparison of HSYA at the same level, GnRHa had stronger inhibition, but in a certain concentration range HSYA inhibited the proliferation of the ECs than GnRHa stronger.
5 Both HSYA and GnRHa inhibited the expression of VEGF and bFGF in cell culture supernatant dose-dependent. GnRHa inhibited the expresstion of VEGF stronger than HSYA. HSYA inhibited the expresstion of bFGF stronger than GnRHa.
6 Both drugs could inhibited normal endometrial cells, had no statistically significant.
HSYA inhibited the proliferation of endometrial cells from EMs patients may be through its down regulation the expression of VEGF and bFGF implementation. Prompted HSYA may be a potential drug for the treatment of endometriosis, to provide a new theoretical basis for the theropy of endometriosis.
引文
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