补肾活血汤对多囊卵巢大鼠的影响
摘要
目的：
本实验成功地建立了多囊卵巢大鼠病理模型，并以此为研究对象，运用光镜、放射免疫、免疫组化及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)技术，观察中药补肾活血汤对多囊卵巢大鼠内分泌、卵巢形态学的影响，重点研究卵巢卵泡闭锁、颗粒细胞凋亡及Bcl-2，Bax的表达情况，以期从细胞及分子水平揭示补肾活血汤的作用机制，为新药的研制和开发提供理论依据。
材料及方法：
选用24日龄未成年雌性SD大鼠60只，随机分为病理模型组50只和正常对照组10只。应用皮下埋植左炔诺孕酮硅胶棒联合皮下注射HCG法建立多囊卵巢动物模型。造模成功后给予补肾活血汤高、中、低剂量灌胃30天，对照组给予同容积蒸馏水灌胃。
于24日龄、36日龄、67日龄测体重，比较造模前后、用药前后大鼠体重。
大鼠断头处死，取血，收集血清样品，放免法测定血清中孕酮(P)、雌二醇(E2)、睾酮(T)的含量。
大鼠处死后立即取出双侧卵巢称重，分别测量卵巢长径(L)、短径(S)，计算平均卵巢面积MOP=L×S、卵巢体积V=4.19×((L+S)／2)~3。
卵巢切片常规HE染色，计算每张切片黄体数、囊状卵泡数、囊状扩张卵泡比例，进行卵泡膜细胞层最大厚度及颗粒细胞层最大厚度的显微测量。
TUNEL法检测颗粒细胞凋亡，以细胞核出现褐色颗粒为阳性结果，计算每张切片凋亡颗粒细胞数、闭锁卵泡数，测量最大闭锁卵泡直径。
采用免疫组织化学SABC法测定卵巢Bcl-2，Bax的表达。以细胞浆内出现棕黄色颗粒为阳性结果。每张切片在400倍高倍视野下随机取5个视野，根据阳性细胞所占比例和染色强度进行半定量判定。染色强度按下列标准评定：阴性为0分；染色虽弱但明显强于阴性对照者为1分；染色强度中等者为2分；染色强者为3分。染色的阳性细胞所占比例评定标准为：阳性细胞数＜5％为阴性(0分)；6％-25％为弱阳性(1分)；26％-50％者为阳性(2分)；51-75％者为强阳性(3分)；＞75％者为非常强阳性(4分)。两种评分相加，即为其阳性积分。
数据处理：计量资料以均值±标准差表示，组间差异的比较采用双侧t检验。率的比较采用两样本率等价比较的u检验。P＜0.05为差别显著，P＜0.01为差别非常显著。
结果：
大鼠体重：补肾活血汤高、中、低剂量组大鼠用药前体重与模型组比较无显著性差异，用药后均明显低于模型组。
卵巢大体解剖观察结果：造模后模型组大鼠卵巢重量、体积、平均卵巢面积均大于对照组(P均＜0.01)，卵巢表面较苍白，见较多囊状扩张的卵泡。补肾活血汤治疗后卵巢色泽红润，高剂量组卵巢重量较模型组(67日龄)明显降低(P＜0.05)，卵巢体积、平均卵巢面积较模型组(36日龄)均明显降低(P均＜0.05)。
卵巢形态学：造模后模型组囊状扩张卵泡比例明显增加，囊状扩张卵泡内卵母细胞或放射冠消失，颗粒细胞层数减少，排列疏松，颗粒细胞层厚度较对照组明显变薄，P＜0.01，泡膜细胞层厚度较对照组明显增厚，P＜0.05，内外卵泡膜细
Objective: The aim of this study was to investigate the effects of Bu Shen Huo Xue Decoction(BSHXT) in rats with polycystic ovary and to explore the possible mechanism of BSHXT in cells and molecules levels. Ovarian morphology, serum hormone, apoptosis of granulosa cells ,atresia of follicles and expression of Bcl-2 and Bax were observed with optic microscope, radio-immunity immunological histo chemistry, terminal deoxynucleotidy transferase-mediated dUTP nick end labeling(TUNEL), and immunohistochemical staining.
Material and method: 24-day-old immature female SD rats (n=60) were separated into two groups at random. One group was PCOS animal model(n= 50), the other group was used as control(n=10). progesterone-synchronized immature rats were used as the animal model for Polycystic ovary (PCO) by injecting 1.5 IU hCG twince daily to 36 days old. And then separated 3 groups (11 rats per group) from the model group, treated with BSHXT for 30 days, separated 7 rats from the model group as the control group.
After treatment, killed the rats. Blood specimens were centrifuged at 3000r /min for 20 minutes, the supernatant was collected for measuring E2 ,P ,T.
Weighed the ovarian weight, measured the ovarian long diameter and short diameter, estimated ovarian volume and mean ovarian product (MOP).
Counted the number of luteum and cystic follicles, measured the thickness of thecal cells and granulosa cells in general HE staining section.
The apoptosis of granulosa cells was checked by TUNEL. Brown drops in nucleolus were regarded as the positive staining. Count the number of apoptosis of granulosa cells and atresia of follicles. Measure the diameter of atresic follicles.
The expressions of Bcl-2 and Bax were checked by immunohistochemical staining. Light brown drops in cells were regarded as the positive staining. Five sights were selected in 400X sight at random, the score of positive cells/all cells ratio and positive cell's staining intensity was counted. Added up the two scores and got the positive integral.
Statistics: Quantitative data were expressed as mean ± SE. Comparison between groups was undertaken using paired t-tests. Percentage data were undertaken using u-tests. P<0.05 was defined as statistically significant.
本实验成功地建立了多囊卵巢大鼠病理模型，并以此为研究对象，运用光镜、放射免疫、免疫组化及末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)技术，观察中药补肾活血汤对多囊卵巢大鼠内分泌、卵巢形态学的影响，重点研究卵巢卵泡闭锁、颗粒细胞凋亡及Bcl-2，Bax的表达情况，以期从细胞及分子水平揭示补肾活血汤的作用机制，为新药的研制和开发提供理论依据。
材料及方法：
选用24日龄未成年雌性SD大鼠60只，随机分为病理模型组50只和正常对照组10只。应用皮下埋植左炔诺孕酮硅胶棒联合皮下注射HCG法建立多囊卵巢动物模型。造模成功后给予补肾活血汤高、中、低剂量灌胃30天，对照组给予同容积蒸馏水灌胃。
于24日龄、36日龄、67日龄测体重，比较造模前后、用药前后大鼠体重。
大鼠断头处死，取血，收集血清样品，放免法测定血清中孕酮(P)、雌二醇(E2)、睾酮(T)的含量。
大鼠处死后立即取出双侧卵巢称重，分别测量卵巢长径(L)、短径(S)，计算平均卵巢面积MOP=L×S、卵巢体积V=4.19×((L+S)／2)~3。
卵巢切片常规HE染色，计算每张切片黄体数、囊状卵泡数、囊状扩张卵泡比例，进行卵泡膜细胞层最大厚度及颗粒细胞层最大厚度的显微测量。
TUNEL法检测颗粒细胞凋亡，以细胞核出现褐色颗粒为阳性结果，计算每张切片凋亡颗粒细胞数、闭锁卵泡数，测量最大闭锁卵泡直径。
采用免疫组织化学SABC法测定卵巢Bcl-2，Bax的表达。以细胞浆内出现棕黄色颗粒为阳性结果。每张切片在400倍高倍视野下随机取5个视野，根据阳性细胞所占比例和染色强度进行半定量判定。染色强度按下列标准评定：阴性为0分；染色虽弱但明显强于阴性对照者为1分；染色强度中等者为2分；染色强者为3分。染色的阳性细胞所占比例评定标准为：阳性细胞数＜5％为阴性(0分)；6％-25％为弱阳性(1分)；26％-50％者为阳性(2分)；51-75％者为强阳性(3分)；＞75％者为非常强阳性(4分)。两种评分相加，即为其阳性积分。
数据处理：计量资料以均值±标准差表示，组间差异的比较采用双侧t检验。率的比较采用两样本率等价比较的u检验。P＜0.05为差别显著，P＜0.01为差别非常显著。
结果：
大鼠体重：补肾活血汤高、中、低剂量组大鼠用药前体重与模型组比较无显著性差异，用药后均明显低于模型组。
卵巢大体解剖观察结果：造模后模型组大鼠卵巢重量、体积、平均卵巢面积均大于对照组(P均＜0.01)，卵巢表面较苍白，见较多囊状扩张的卵泡。补肾活血汤治疗后卵巢色泽红润，高剂量组卵巢重量较模型组(67日龄)明显降低(P＜0.05)，卵巢体积、平均卵巢面积较模型组(36日龄)均明显降低(P均＜0.05)。
卵巢形态学：造模后模型组囊状扩张卵泡比例明显增加，囊状扩张卵泡内卵母细胞或放射冠消失，颗粒细胞层数减少，排列疏松，颗粒细胞层厚度较对照组明显变薄，P＜0.01，泡膜细胞层厚度较对照组明显增厚，P＜0.05，内外卵泡膜细
Objective: The aim of this study was to investigate the effects of Bu Shen Huo Xue Decoction(BSHXT) in rats with polycystic ovary and to explore the possible mechanism of BSHXT in cells and molecules levels. Ovarian morphology, serum hormone, apoptosis of granulosa cells ,atresia of follicles and expression of Bcl-2 and Bax were observed with optic microscope, radio-immunity immunological histo chemistry, terminal deoxynucleotidy transferase-mediated dUTP nick end labeling(TUNEL), and immunohistochemical staining.
Material and method: 24-day-old immature female SD rats (n=60) were separated into two groups at random. One group was PCOS animal model(n= 50), the other group was used as control(n=10). progesterone-synchronized immature rats were used as the animal model for Polycystic ovary (PCO) by injecting 1.5 IU hCG twince daily to 36 days old. And then separated 3 groups (11 rats per group) from the model group, treated with BSHXT for 30 days, separated 7 rats from the model group as the control group.
After treatment, killed the rats. Blood specimens were centrifuged at 3000r /min for 20 minutes, the supernatant was collected for measuring E2 ,P ,T.
Weighed the ovarian weight, measured the ovarian long diameter and short diameter, estimated ovarian volume and mean ovarian product (MOP).
Counted the number of luteum and cystic follicles, measured the thickness of thecal cells and granulosa cells in general HE staining section.
The apoptosis of granulosa cells was checked by TUNEL. Brown drops in nucleolus were regarded as the positive staining. Count the number of apoptosis of granulosa cells and atresia of follicles. Measure the diameter of atresic follicles.
The expressions of Bcl-2 and Bax were checked by immunohistochemical staining. Light brown drops in cells were regarded as the positive staining. Five sights were selected in 400X sight at random, the score of positive cells/all cells ratio and positive cell's staining intensity was counted. Added up the two scores and got the positive integral.
Statistics: Quantitative data were expressed as mean ± SE. Comparison between groups was undertaken using paired t-tests. Percentage data were undertaken using u-tests. P<0.05 was defined as statistically significant.
引文
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9．俞瑾．多囊卵巢综合征的中西医治疗．中国实用妇科与产科杂志，2002，18(11)：651～653．
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12．郝兰枝冲药人工周期疗法治疗多囊卵巢综合征中高雄激素血症的临床观察．山东中医杂志，2004，23(8)：467～468．
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14．许金榜，林莺，吕绍光．健脾益肾活血法改善多囊卵巢综合征胰岛素抵抗理论探讨．福建中医学院学报，2005，15(2)：50～51．
15．郝琦蓉，张引华．多囊平胶囊治疗多囊卵巢综合征的实验研究．山西医科大学学报，2003，34(5)：447～450．
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2．王太平，王淑艳，于晓．二甲双胍治疗多囊卵巢综合征的研究进展．中华临床医药，2003，4(7)：63～64．
3．李云波，张丽梅，侯丽辉．中西医结合治疗多囊卵巢综合征的临床和实验研究进展．中医药信息，2004，21(1)：11～12．
4．张勉之，张大宁．补肾活血法浅析．长春中医学院学报，2003，19(4)：6～7．
5．尤昭玲，杨正望，傅灵梅．多囊卵巢综合征从肾虚血瘀调治的探讨．湖南中医学院学报，2005，25(1)：25～26．
6．赵珂，王东梅．多囊卵巢综合征中医证候研究．2004，14．
7．许小凤，王迎．多囊卵巢综合征基础性激素水平与中医辨证的相关性．江苏中医药，2004，25(11)：14～16．
8．候璟玟，俞瑕，魏美娟．中药天癸方治疗多囊卵巢综合征中高雄激素高胰岛素血症的研究．中国中西医结合杂志，2000，20(8)：589～592．
9．俞瑾．多囊卵巢综合征的中西医治疗．中国实用妇科与产科杂志，2002，18(11)：651～653．
10．俞瑾．功能性月经失调的中西医结合疗效标准(试行草案)．中国中西医结合杂志，2003，(3)：237～239．
11．王兴娟．承师心悟—滋阴降火法治疗多囊卵巢综合征．上海中医药杂志，1999，(12)：22～23．
12．郝兰枝冲药人工周期疗法治疗多囊卵巢综合征中高雄激素血症的临床观察．山东中医杂志，2004，23(8)：467～468．
13．叶一萍．辨病与辨证结合治疗多囊卵巢综合征．辽宁中医学院学报，2004，6(4)：310．
14．许金榜，林莺，吕绍光．健脾益肾活血法改善多囊卵巢综合征胰岛素抵抗理论探讨．福建中医学院学报，2005，15(2)：50～51．
15．郝琦蓉，张引华．多囊平胶囊治疗多囊卵巢综合征的实验研究．山西医科大学学报，2003，34(5)：447～450．
16．孙永生．卵巢Ⅱ号对多囊卵巢大鼠卵巢形态学的影响．中医药信息，2002，19 (3)：70～71．
17．孙斐，俞瑾．雄激素致不孕大鼠无排卵机制及滋肾阴药作用的探讨．中华内分泌代谢杂志，1999，15(5)：259～262．
18．张月萍，俞瑾，归绥琪．雄激素致不孕大鼠发病机制及滋肾阴药对其促排卵作用．中华内分泌代谢杂志，1994，(10)：98～101．
19．俞瑾．补肾化痰治疗多囊卵巢综合征中对下丘脑—垂体—卵巢功能的调节．中西医结合杂志，1986，(4)：218～221．
20．刘嘉．马丽萍等．滋阴疏肝汤对多囊卵巢小鼠内分泌功能的影响．中国中西医结合皮肤性病学杂志，2005，4(1)：29～30．