摘要
子宫内膜异位症(内异症)是一种雌激素依赖的慢性妇科疾病,在生育期妇女发中发病率达10-15%,主要临床表现为盆腔痛和不孕,但病因不明。目前得到广泛接受的是种植学说,即内异症是由经血逆流或通过淋巴、静脉种植到子宫内膜以外的地方生长。但经期腹腔镜手术证实,76-90%妇女发生经血逆流,远高于内异症发病率,表明经血种植常常发生但少部分妇女罹患内异症,因此种植学说不能完全解释内异症病因。近十余年来,关于子宫内膜和内异灶中存在干细胞、干细胞在内异症病因学方面的证据表明,内异症可能是一种干细胞相关疾病。子宫内膜中的干细胞可能随经血种植并内异症形成和发展中起重要作用,因此干细胞学说可以看作是种植学说的一种延伸。
研究表明骨髓来源的干细胞可在体内分化为在位和异位子宫内膜细胞,干细胞分化为异位子宫内膜可能是内异症的一种新的发病机制。2004年,Taylor, H.S.等对4名接受HLA单抗原不匹配骨髓移植、具有子宫内膜活检适应症的妇女进行了子宫内膜活检,通过免疫组化和RT-PCR法检测HLA,发现受体的子宫内膜活检组织中有0.2%-48%的上皮细胞和0.3%-52%的间质细胞来源于供体,首次提出受体的子宫内膜细胞可以来源于供体的骨髓细胞,提示非子宫部位的干细胞可能对子宫内膜的再生起一定作用。2007年,Du,H.等将雄鼠骨髓细胞通过尾静脉注入放射线照射后的雌鼠血液中,6个月后在雌鼠子宫内膜中找到了雄鼠来源的骨髓细胞,同时通过给切除子宫的LacZ雌鼠注射野生型雌鼠的子宫组织碎片进行内异症造模,10周后在异位内膜中发现表达LacZ的子宫内膜上皮细胞和间质细胞。尽管来源于供体骨髓细胞的上皮和间质细胞所占比例很小,但提示了血液循环中骨髓来源的干细胞可能具有分化为布位和异位子宫内膜细胞的潜能。
本课题组前期将转基因红色荧光蛋白雄鼠骨髓移植到雌鼠体内,发现雌鼠子宫腺上皮与间质中均有雄鼠来源的骨髓细胞,与Du,H.等骨髓来源的干细胞可在体内分化为子宫内膜细胞的结论一致。骨髓来源的干细胞包括造血干细胞和间充质干细胞两大类,前者可分化为白细胞、红细胞等各符类血细胞,后者向中胚层方向骨、软骨、肌肉、脂肪等各类组织分化。组织胚胎学方面,子宫完全发育自中胚层的副中肾管,包括子宫内膜腺上皮、间质细胞和所有的纤维肌肉组织,与食道、胃等一般脏器上皮来自内胚层、纤维肌肉组织来自中胚层完全不同。因此我们设想子宫内膜腺上皮来源于骨髓来源干细胞中的间充质干细胞,若体外能够将骨髓间充质干细胞诱导为子宫内膜腺上皮,将具有极大的临床治疗意义。
在此基础上,课题组开展了体外诱导骨髓间充质干细胞(BMSCs)向子宫内膜腺上皮方向分化的研究,体外将BMSCs与子宫内膜间质细胞(EStCs)间接共培养,realtime RT-PCR检测上皮方面标记物角蛋白7、18、19、上皮膜抗原显著增加,免疫荧光进一步验证共培养后角蛋白显著增加,表明骨髓间充质干细胞在体外共培养体系下可以向子宫内膜腺上皮方向分化。为进一步研究BMSCs向子宫内膜腺上皮方向分化过程发生的一系列重要蛋白质分子及信号转导通路变化,故对诱导前后的BMSCs、子宫内膜腺上皮行同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation, iTRAQ)蛋白质组学分析,以获得这一分化过程的重要蛋白分子及信号通路变化信息。本课题具体分为以下三部分。
第一部分,为获取足量的未分化、分化BMSCs、子宫内膜腺上皮以行蛋白质组学分析、比较,我们原代培养并鉴定小鼠BMSCs和EStCs,建立了两种细胞的共培养体系促使BMSCs向子宫内膜腺上皮分化,并采用激光捕获显微切割技术获取足量小鼠子宫内膜腺上皮,提取五组样品的蛋白,调整五组蛋白量后上机行iTRAQ蛋白质组学检测,并通过数据库查找信息,明确208种蛋白名称及其相对定量,为探寻BMSCs向子宫内膜腺上皮方向分化的差异蛋白分子及相关信号通路分析提供坚实的基础。
第二部分,选取蛋白质组学鉴定结果中上皮细胞和间质细胞标记进行western blot检测,在对蛋白质组学鉴定结果进行验证的同时,进一步说明BMSCs向子宫内膜腺上皮方向分化。验证结果表明大部分组学结果可靠,并通过进一步验证随共培养时间增加上皮细胞标志物表达增加、间质细胞标记物表达下降,进一步阐明了骨髓间充质干细胞在共培养体系下随时间进展不断向子宫内膜方向分化。
第三部分,为研究BMSCs向子宫内膜腺上皮方向分化过程发生的一系列蛋白质分子及信号转导通路变化,选取第七天共培养诱导分化组与单独培养组比值>1.2或<0.833的蛋白作为差异蛋白进行分析。差异蛋白的系统聚类分析从整体差异蛋白水平进一步阐明了共培养体系诱导后的分化；差异蛋白的分子功能、生物学过程、信号通路的分析显示,能量代谢是重要基础,而分化细胞的形态形成、运动、粘连和发育均在此基础上实现；Nes、Sod2、Hsp90、Anxal等差异蛋白质分子直接或间接通过a-catenin、NF-kB等转录因子直接或间接调控Wnt、MAPK/ERK、PI3K/Akt等信号通路实现BMSCs向子宫内膜腺上皮方向的分化。
综上所述,在小鼠骨髓间充质干细胞向子宫内膜方向分化的蛋白质组学研究中,我们从上皮细胞和间质细胞标记的蛋白个体和差异蛋白整体水平均进一步阐明了共培养体系下BMSCs不断向子宫内膜腺上皮方向分化；差异蛋白质分子直接或间接通过α-catenin、NF-κB等转录因子直接或间接调控Wnt、MAPK/ERK、PI3K/Akt等信号通路实现BMSCs向子宫内膜腺上皮方向的分化,而调控差异蛋白质分子及信号通路可能对临床上治疗内异症等子宫内膜相关疾病具有一定的意义。
绒癌是一种罕见的恶性滋养细胞肿瘤,一项美国大规模流行病学调查显示,每10,000妇女年有0.133例为绒癌。绝大多数绒癌原发于子宫体,但也有极少数可原发于输卵管、宫颈、阔韧带等部位。目前,绒癌的病因学机制仍不明。不明部位持续性妊娠(PPUL)是指患者血HCG异常升高,血HCG水平在<500mIU/mL或<2,000mIU/mL范围内的处于平台期或持续性升高,但经阴道超声检查(TVS)始终在宫内和宫外发现妊娠证据。[2-4]本论文报道PPUL确诊异位绒癌罕见疑难病例一例,并抽取患者静脉血提取基因组DNA行肿瘤相关基因TP53和ERBB2外显子测序分析,在此基础上发现具有致病性可能的TP53SNPrs1042522表现为C/G杂合型,由此展开TP53单核苷酸多态性rs1042522与绒癌的相关性研究。
第一部分,我们报道异位绒癌罕见疑难病例具体诊疗经过以及该患者TP53和ERBB2基因外显子测序分析结果。TVS始终在宫内和宫外发现妊娠证据,经过两次全身甲氨喋呤(MTX)治疗血HCG仍升高,电视腹腔镜手术、刮宫术及术后病理均未能发现输卵管或卵巢病灶,后在胸片提示左肺下叶见结节下考虑到诊断为绒癌,随后在盆腔、胸部、颅脑MRI辅助检查下,临床诊断为异位绒癌,经化疗和电视胸腔镜切除转移病灶手术联合治疗及时消除原发灶和转移灶。在进行目的基因外显子测序中,ERBB2外显子20中第116位碱基发生杂合性同义突变(G>A),蛋白质序列并未改变；而具有致病性可能的TP53SNP rs1042522表现为C/G杂合型,并由此推测该SNP精氨酸脯氨酸杂合状态可能是患者具有绒癌易感性的基础。
第二部分,我们在前期基础上进一步展开了rs1042522与绒癌的相关性研究。我们抽取绒癌组23名、对照组84名的静脉血5mL对rs1042522进行测序分析,Hardy-Weinberg平衡检验结果显示入组人群符合遗传平衡状态,结果显示无论是等位基因C相比G还是基因型C/C、C/G、C/G+C/C相比G/G,其相对危险度OR值均大于1,但差异无统计学意义,提示rs1042522与绒癌之间可能并不一定存在相关关系。我们推测,一种情况是rs1042522等位基因C相对的p53第72位氨基酸脯氨酸可以增加绒癌易感性,但是受绒癌发病率低、目前绒癌组样本量小的影响,差异无统计学意义；另一种情况是rs1042522与绒癌的易感性之间并不存在关系。因此,在目前工作基础上,下一步课题组需继续扩大样本量进行研究。
综上,课题组在PPUL确诊异位绒癌罕见疑难病例基础上展开TP53单核苷酸多态性rs1042522与绒癌的相关性研究,目前研究结果提示rs1042522与绒癌之间并不一定存在相关关系,但可能受到绒癌组人数少的影响,需继续扩大绒癌组样本量进行研究。
Objective:Male mice bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our group has demonstrated that BMSCs can differentiate in the direction of EECs in condition of co-culture with endometrial stromal cells in vitro. Here we tend to obtain information in the process of differentiation of BMSCs into EEC by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis and discern some potential pathways in the process.
Methods:A0.4μm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in Transwell chamber into BMSCs. After indirect co-culture for several days, BMSCs were revealed to progressively differentiate towards epithelial like cells in vitro. Then five groups were divided according to different co-culture days with EECs and BMSCs single culture groups as control. Differential candidate proteins were analysed through proteomic approach of based iTRAQ quantitative differential LC-MS/MS. Four proteins were verified by Western blot. Gene ontology (GO) analysis and pathway analysis were applied.
Results:A total number of208proteins were identified,100of which proteins were differentially expressed with1.2-fold difference, with52up-regulated and48proteins down-regulated. Four proteins verified by Western blot were in general accordance with the results of iTRAQ proteomics. Gene ontology analysis of differential proteins revealed that cellular morphogenesis, motility, adhesion and development of differentiated cells are based on the metabolic process. Ingenuity IPA software analysis demonstrated differential proteins such as Nes, Sod2、Krt8、HSP90、 Anxal play a potential important role in promoting BMSCs to differentiate into EEC by directly and indirectly regulating signaling pathway including NF-κB, MAPK/ERK and P13K/Akt etc.
Conclusion:Our study demonstrated the detailed alteration of proteins and signaling pathways during mice BMSCs differentiating into EECs in vitro. More mature autologous EECs could be induced from BMSCs by targetting these important proteins and signaling pathways, which was of potential clinical significance in providing new treatment for Asherman syndrome and endometriosis.
Choriocarcinoma is a kind of malignant gestational trophoblastic neoplasia with low incidence. The incidence of choriocarcinoma is estimated to be0.133per100,000woman-years.[l] Most choriocarcinoma lesions located in uterus, however, there were extremely few primary lesions locating in fallopian tubes, cervix or broad ligment etc. The etiology of choriocarcinoma is still unkown to date. Persisting pregnancy of unknown location means women have a plateau or increase in serial serum HCG concentrationsless than500mlU/mL or less than2,000mIU/mL, but no evidence of intrauterine or ectopic pregancy can be found by transvaginal sonography.[2-4] In this article, we present information concerning a rare ectopic choriocarcinoma characterized by invisible primary lesions and low levels of serum human chorionic gonadotropin (hCG) that masqueraded as a persisting pregnancy of unknown location (PPUL). Genomic DNA was extracted from peripheral blood leukocytes of the patient. All ERBB2and P53coding exons were amplified by polymerase chain reaction and subjected to automatic DNA sequencing. In TP53, a SNP rs1042522in codon72(C/G at position119in exon4) was identified. On this basis, a study of SNP rs1042522and susceptiblity of choriocarcinoma was performed.
In the first part, we reported the the rare ectopic choriocarcinoma characterized by invisible primary lesions and low levels of serum HCG that masqueraded as a PPUL and the DNA sequencing results of all ERBB2and P53coding exons. No evidence of intrauterine or ectopic pregancy had been found by transvaginal sonography and serum HCG still increased in case of MTX therapy twice. TV assisted laparoscopy, uterine curretage and pathology examination provided no evidence of lesion. It was not until the chest X-ray revealed a nodule in the left lower lobe that we considered the possibility of choriocarcinoma. Pelvic, abdominal and cranial MRI were used to additionally detect a primary left adnexal lesion and a metastatic nodule of choriocarcinoma in the left lower lobe. To our knowledge, this case was the first case of ectopic choriocarcinoma masquerading as PPUL in which the primary and metastatic lesions were timely treated by a combination of chemotherapy and surgery before systemic metastasis. The results of DNA sequencing showed a heterozygous synonymous mutation in codon793(G>A at position116in exon20) in ERBB2and the protein codon did not change. In TP53, a SNP rs1042522in codon72(C/G at position119in exon4) was identified. TP53rs1042522with probablepathogenic allele in Reference SNP Cluster Report, so we hypothesized that rs1042522C/G heterozygosity might be the genetic basis of choriocarcinoma susceptibility.
In the second part, we further performed the study of rs1042522and susceptibility of choriocarcinoma on previous basis. Genomic DNA were extracted from peripheral blood leukocytes of23choriocarcinoma patients and84healthy women as control to sequence rs1042522. The chosen population fit Hardy-Weinberg equilibrium. Both the odds ratio (OR) of allele C compared to allele G and the OR of genotype C/C, C/G, C/G+C/C compared to G/G were larger than1, but the differences are of no significance, which suggested rs1042522might not correlate with susceptibility of choriocarcinoma. We presumed the small number of choriocarcinoma patients might affect the statistic analysis. Thus, more patients should be enrolled to explore the rs1042522and susceptibility of choriocarcinoma.
In summary, we performed the study of rs1042522and susceptibility of choriocarcinoma on basis of the rare ectopic choriocarcinoma masqueraded as a PPUL. The present result suggested rs1042522might not correlate with susceptibility of choriocarcinoma, but might be affected by the small number of choriocarcinoma patients. Therefore, more patients will be enrolled to continue exploring the study.
引文
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