【作者】 王玉珏；

【导师】 顾为望；

【作者基本信息】 南方医科大学 ， 动物学， 2008， 硕士

【摘要】 随着对肿瘤放疗领域研究的不断深入,肿瘤患者预后明显改善,患者生存率明显提高。据美国1995年～2005年间数据统计,所有诊断浸润癌患者（无论年龄大小）5年生存率接近60%。其中年轻女性肿瘤患者5年生存率远高于60%,治疗所致的医源性损害已逐渐得到重视。如放疗常见的一个并发症卵巢毒性引起的早绝经和不育严重影响患者的生活质量和自信。近年有关放疗对卵巢损害的机制以及预防卵巢受损的研究受到了肿瘤学、生殖医学、放射防护学等学科的关注,本文就利用骨髓细胞的发育潜能来修复放疗损伤卵巢组织的可能性及其机制进行了初步实验研究。本研究的目的是通过观察骨髓移植对放疗引起的卵巢受损小鼠的修复效果,比较同性/异性骨髓移植对放射引起的卵巢损伤修复的差异,以及推测其损伤修复机制。观察的指标包括:血清中FSH、E₂含量,卵巢系数（OI）,卵巢组织的组织形态及是否出现动情周期。本论文分为三个部分。第一部分为研究骨髓移植是否对放疗引起的小鼠卵巢损伤有恢复作用;第二部分为比较同性或异性骨髓移植对放疗引起的小鼠卵巢损伤的修复是否有差异;第三部分为关于放疗损伤卵巢组织功能恢复机制的研究。各部分的实验方法如下:1.骨髓移植对放疗引起的小鼠卵巢损伤修复作用的实验中,我们采用140只SPF级8～10周龄雌性昆明小鼠,除20只作为骨髓移植供体,其余120只随机分为平均三组:正常对照组,单次放疗组,骨髓移植组。正常对照组,不采取任何措施;单次放疗组,单次放疗后不采取任何措施;骨髓移植组,单次放疗后24h进行骨髓移植。骨髓移植后于第1,5,15,30天四个时间点分别从各组中取10只小鼠检测血清中E₂,FSH水平并测定OI,取卵巢组织做病理切变观察;采用析因方差方法进行统计。2.同性或异性骨髓移植对放疗引起的小鼠卵巢损伤的修复实验中,我们采用175只SPF级8～10周龄雌性昆明小鼠,15只SPF级8～10周龄雄性昆明小鼠。其中15只雄性小鼠作为雄性骨髓移植供体,15只雌性小鼠作为雌性骨髓移植供体。余下160只雌性小鼠随机分为四组:正常对照组,单次放疗组,同性骨髓移植组,异性骨髓移植组。正常对照组,不采取任何措施;单次放疗组,单次放疗后不采取任何措施;异性骨髓移植组,单次放疗后24h内移植雄性小鼠骨髓细胞;同性骨髓移植组,单次放疗后24h内移植雌性小鼠骨髓细胞。于骨髓移植后第1,5,15,30天每个时间点分别从各组中取10只小鼠检测血清中E₂,FSH水平,并测定OI,并取卵巢组织做病理切变观察;采用析因方差方法进行统计。3.骨髓细胞移植到放疗后小鼠的体内实验中,我们采用了22只雌性ICR小鼠,其中10只作为荧光骨髓移植受体,6只作为单次放疗组,6只作为空白对照组。另有5只带有绿色荧光标记的转基因ICR小鼠作为骨髓移植供体。单次放疗组单次放疗后不采取任何措施;空白对照组,不采取任何措施;骨髓移植组,单次放疗后24h移植荧光小鼠骨髓细胞。骨髓移植后的第15,30天分别取对照组3只,单次放疗组3只,荧光骨髓移植组5只小鼠的卵巢组织做冰冻切变观察是否出现带有荧光标记的卵细胞。实验结果如下:1.放射后放疗后骨髓移植组（E₂:10.83ng/L,FSH:2.94IU/L,OI:2.16）,单次放疗组（E₂10.30ng/L,FSH2.84IU/L,OI:2.24）的激素水平、卵巢系数与正常组（E₂10.83ng/L,FSH2.42IU/L,OI:2.70）差异显著,在放疗后第15、30天,单次放疗组成熟卵泡数量减少,卵巢血管增生,有纤维化;骨髓移植组可见原始卵泡,初级卵泡,次级卵泡,成熟卵泡,血管较丰富。血清中E₂、FSH水平及OI在放疗后第15、30天骨髓移植组（E₂23.02ng/L,FSH2.59IU/L,OI:2.60;E₂23.92ng/L,FSH2.24IU/L,OI:2.76）与单次放疗组（E₂18.17ng/L,FSH2.98IU/L,OI:2.34;E₂19.62ng/L,FSH3.05IU/L,OI:2.47）有显著性差异。（血清中E₂、FSH水平及卵巢系数在第15天时的F值分别为15.676,2.854,27.215;P值分别为0.000,0.000,0.045;第30天时的F值分别为12.610,15.657,19.709;P值分别为0.000,0.000,0.000）。2.放疗后初期同性骨髓移植组、异性骨髓移植组、单次放疗组的E₂,FSH,OI分别为（12.12ng/L,3.59IU/L,2.23;12.10ng/L,3.56IU/L,2.27;10.38ng/L,3.29IU/L,2.22）,与正常对照组（25.35ng/L,2.36IU/L,2.83）差异显著;在骨髓移植第15、30天,单次放疗组、异性骨髓移植组成熟卵泡数量持续减少,卵巢血管增生,有纤维化,较实验初期未见明显好转;同性骨髓移植组可见原始卵泡,初级卵泡,次级卵泡,成熟卵泡,血管较丰富。在第15天同性骨髓移植组（E₂18.38ng/L,FSH3.10IU/L,OI:2.59）血清中E₂、FSH水平及OI与单次放疗组（E₂10.83ng/L,FSH2.42IU/L,OI:2.70）、异性移植组（E₂12.54ng/L,FSH3.88IU/L,OI:2.46）有显著性差异,与正常对照组（E₂25.39ng/L,FSH2.39IU/L,OI:2.82）接近,并可以继续好转至30日。（血清中E₂、FSH水平及OI在第15天时的F值分别为124.038、95.431、15.346;P值分别为0.000、0.000、0.000;在第30天时的F值分别为180.967、190.495、10.968;P值分别为0.000、0.000、0.000）3.骨髓移植后第15,30天,冰冻切片观察各组卵巢内卵细胞生长情况时发现,移植荧光小鼠骨髓的雌性小鼠卵巢中均发现不同程度的荧光细胞,而对照组别未发现荧光细胞。通过以上研究,我们可以判断出骨髓移植对由放疗引起的小鼠卵巢损伤具有一定恢复作用,但是只有同性之间的骨髓移植才对卵巢恢复起作用,同时证明了外源性的骨髓干细胞不仅可以促进小鼠本身卵巢功能的恢复,而且可以在放疗后小鼠卵巢中继续发育成为卵细胞。更多还原

【Abstract】 With rapid development in the field of tumor radiotherapy,the fidelity of prognosis and surviving rate of patients with cancer improved obviously.According to the statistical results from 1995～2005 in U.S.A,the survive rate of the infiltrating carcinoma patients reached as high as 60%.With the improvement of long term survive rate of young female patients,more and more attention was paid to iatrogenic harm.For example,long term physiological and psychological hurt.One of long term complication in radiotherapy is ovary toxicity.This is due to the finity of ovary germ cells,which can’t be regenerated.Therefore,to many young female patients who expect normal pregnant,the sterility and early menopause caused by radiotherapy affect their life quality and self-confidence seriously.Recently,the study on the mechanisms and prevention of ovary toxicity caused by radiotherapy aroused the interest of researchers in the field of phymatology,reproductive medicine,radiative prevention.This paper initially studied the feasibility and mechanisms of the recovery of radiotherapic ovary damage by marrow transplantation.The aim of this paper is to determine the recovery level of radiotherapic ovaries after marrow transplantation by observing pathological morphous,testing FSH,E₂ level in blood serum of mouse and checking whether estrous cycle was reoccurred. Then,after observing the differences between homogeneity marrow transplantation group（HOMT） and heterosexual（HEMT） marrow transplantation groupto,a new mechanism for ovary recovery can be provided. The study was divided into three parts.The first part mainly studied the fuction of ovary recovery by transplanting marrow into radiotherapic mouse;the second part discussed different ovary recovery levels between HOMT and HEMT;the third part initially explained the possible mechanism of recovery in this way.1.In the first experiment,we used 140 8～10 weeks（w） Kunming female mouse（SPF,same in the following experiments）,20 as transplant donor,40 as control group which didn’t receive any treatment;40 as radiotherapy group which were radiated once;40 as transplant group which were performed marrow transplant within 24h after radiation.On the day 1^st,5^th,15^th,30^th we selected 10 mouse from each groups respectively to detected the E₂,FSH in blood serum,weighted their ovary index and made pathological section of ovary.2.In the second experiment,we used 175 SPF 8～10w Kunming female mouse and 15 SPF 8～10w male Kunming mouse.15 male mouse as transplant donor and 15 female mouse as transplant donor,40 as control group which didn’t adopt any treatment;40 as radiotherapy group which radiated once only;40 as transplant group which performed female marrow transplant within 24h after radiation,40 as transplant group which performed male marrow transplant within 24h after radiation. On the day 1^st,5^th,15^th,30^th we selected 10 mouse from each groups respectively to detected the E₂,FSH in blood serum and weighted ovary index and made pathological section of ovary.3.In the third experiment,we used 22 female ICR mouse,5 female ICR fluorescent mouse.6 ICR mouse as radiotherapy group which radiated only once,6 ICR mouse as control group which didn’t have any treatment,5 fluoresent mouse as marrow transplant donor,10 ICR as marrow transplant group which transplant fluorescent marrow within 24h after radiation.On day 15^th,30^th,we select half of the group perpform ovary frozen section respectively. In the experiment on whether marrow transplant is good for ovary recovery harmed by radiotherapy,on day 15^th and 30^th,we observed marrow transplant group reappeared more primordial follicle,primary follicle,secondary follicle and mature follicle compared with radiotherapy groupand,and the vessels in ovary was comparatively abundant.The number of ovum in radio therapy group was dropped dramatically.The E₂ and FSH level in blood serum and ovary index between radiotherapy group and marrow transplant group has significant differences on day 15^th and 30^th.(The F value of E₂,FSH,ovary index on 15^th day was 15.676,2.854, 27.215 respectively,the P value of E₂,FSH,ovary index was 0.000,0.000,0.045 respectively;The F value of E₂,FSH,ovary index on 30^th day was 12.610,15.657, 19.709 respectively,the P value of E₂,FSH,ovary index was 0.000,0.000,0.000).In the experiment on whether marrow from different sex is different for ovary recovery harmed by radiotherapy,on day 15^th and 30^th,compared with radiotherapy group,we observed female marrow transplant group reappeared primordial follicle, primary follicle,secondary follicle and mature follicle,the vessels in ovary was comparatively was abundant.The number of ovum in radiotherapy group and male marrow transplant group was dropped dramatically.The E₂ and FSH level in blood serum and OI between radiotherapy group,HEMT and HOMT had significant differences on day 15^th and 30^th.(The F value of E₂,FSH,OI on 15^th day was 124.038,95.431,15.346 respectively,the P value of E₂,FSH,ovary index was0.000, 0.000,0.000 respectively;The F value of E₂,FSH,OI on 30^th day was 180967,190.495,10.968 respectively,the P value of E₂,FSH,ovary index was0.000, 0.000,0.000)In the third part,we found GFP cells in the GFP marrow transplantation group on d15 and d30,while in other groups we didn’t found any GFP cells.According to the results above,a conclustion can be drawed that marrow transplantion of same sex contributes to the recovery of ovary harmed by radiation. The marrow cells from extrinsic sourc have effect on both ovary itself and can be further developed into ovums.更多还原