三种卵巢早衰大鼠造模方法比较研究

Comparative Study on Three Modeling Methods of Premature Ovarian Failure Rats

目的探讨建立简洁稳定高效的卵巢早衰(POF)的大鼠模型。方法将40只具有规律动情周期的SD大鼠按随机数字表法分为正常组、环磷酰胺(CTX)组、二氧化乙烯基环己烯(VCD)组、雷公藤(TG)组,每组10只。大鼠适应性喂养1周后,分别采用CTX、VCD、TG造模。在造模过程中观察大鼠的一般情况和动情周期,每周检测大鼠体重,采用HE染色在光镜下观察卵巢组织形态变化;采用TUNEL法检测卵巢颗粒细胞凋亡情况;采用ELISA检测大鼠血清促卵泡激素(FSH)、雌二醇(E)含量;采用实时荧光定量PCR法(RT-PCR)和Western Blot检测卵巢组织B细胞淋巴瘤-2(Bcl-2)、促凋亡蛋白Bcl-2相关X蛋白(Bcl-2 assaciated X protein,Bax)mRNA及蛋白表达水平。结果与正常组比较,各模型组大鼠出现动情周期紊乱,卵巢组织缺血苍白伴不同程度的萎缩,卵泡数减少、卵巢颗粒细胞凋亡率均升高(P<0.05,P<0.01),血清FSH含量(除TG组外)均明显升高(P<0.01),血清E2含量均明显降低(P<0.01),Bcl-2/Bax mRNA和蛋白表达均降低(P<0.05,P<0.01);与VCD、TG组比较,CTX组大鼠体重减轻(P<0.05),成熟卵泡率明显减少(P<0.01),FSH含量显著降低(P<0.05)。结论CTX诱导建立POF大鼠模型,造模方法简便易行,成功率较高,造模效果优于VCD和TG,是更为理想的POF造模方法。

Objective To observe the establishment of a simple, stable and efficient rat model of premature ovarian failure(POF). Methods Forty SD rats with regular estrous cycles were divided into normal group, cyclophosphamide(CTX) group, vinylcyclohexene dioxide(VCD) group, and Tripterygium wilfordii(TG) group according to random number table,10 in each group. After 1 week of adaptive feeding, the rats were modeled with CTX, VCD, and TG. During the modeling process, the general condition and estrous cycle of the rats were observed. The body weight of the rats was detected every week. The morphological changes of ovarian tissue were observed by HE staining under light microscope. The apoptosis of ovarian granulosa cells was detected by TUNEL method. Serum levels of follicle-stimulating hormone(FSH) and estradiol(E) in rats were detected by ELISA. Real-time quantitative PCR(RT-PCR) and Western Blot were used to detect mRNA and protein expression levels of B-cell lymphoma-2(Bcl-2) and Bcl-2 associated X protein(Bax). Results Compared with normal group, estrous cycle disorder, ovarian tissue ischemia and pallor with different degrees of atrophy, decreased follicle number, increased ovarian granulosa cell apoptosis rate(P<0.05, P<0.01), serum FSH content significantly increased(except TG group)(P<0.01), and significantly decreased serum Econtent(P<0.01), decreased mRNA and protein expressions of Bcl-2/Bax(P<0.05, P<0.01) occurred in each model group. Compared with VCD and TG groups, the body weight of rats was reduced(P<0.05), the rate of mature follicles significantly decreased(P<0.01), and the content of FSH was significantly lowered(P<0.05) in CTX group. Conclusions CTX was a simple and easy method to establish rat model of POF with higher success rate. The modeling effect of CTX was better than that of VCD and TG and became a more ideal method of POF modeling.