Frizzed家族基因mRNA在小鼠动情周期卵巢组织中的表达规律

运用Real-time Q-PCR方法分析了Frizzed1-10mRNA在小鼠动情周期卵巢中的相对表达水平.结果发现10个Frizzed家族基因中,除Frizzed8外,其他均有不同水平的表达.其中Frizzed1和2在动情前期表达水平显著高于其他3个时期水平,约是间情期水平的4倍(P<0.05);动情前期和间情期的Frizzed3表达水平约是另2个时期的1.5倍(P<0.05);Frizzed4在动情期表达水平显著高于其他3个时期,约是间情期的3倍(P<0.05);间情期Frizzed5表达水平显著高于动情前期和动情期,约是动情期的5倍(P<0.05);Frizzed7表达类似于Frizzed5,间情期表达水平约是动情前期的4倍(P<0.05);Frizzed10的表达在动情前期表达水平约是动情期的3倍(P<0.05);Frizzed6和9在4个时期的表达无显著差别(P<0.05).以上结果表明:Frizzeds在4个动情时期卵巢中广泛而差异表达,并推测Frizzed1—4可能在动情周期卵泡发育过程中具有重要作用.     

青春期多囊卵巢综合征患者代谢综合征的患病特点

目的探索青春期多囊卵巢综合征（PCOS）患者代谢综合征（MS）患病特点。方法收集80例青春期PCOS患者MS患病率与肥胖、年龄、胰岛素抵抗和ApoB/ApoA1比值的关系。结果青春期PCOS患者MS患病率为12.50%,其中非肥胖患者MS和前代谢综合征阶段（pre-MS）患病率分别为0.00%和7.55%,低于肥胖患者的37.04%和33.33%;两组pre-MS患病率差异有统计学意义（P＜0.05）。13~15岁组MS患病率为6.67%,低于16~19岁组的13.85%（P＜0.05）;pre-MS患病率为20.00%,高于16~19岁组的15.38%。胰岛素抵抗型患者的MS和pre-MS患病率分别为33.33%和33.33%,高于非胰岛素抵抗型患者的6.45%和11.29%（P＜0.05）,ApoB/ApoA1≥0.6的患者MS和pre-MS患病率分别为33.33%和16.67%,高于ApoB/ApoA1＜0.6的患者6.45%和16.13%（P＜0.05）。结论青春期PCOS患者的MS患病率以肥胖、胰岛素抵抗型、ApoB/ApoA1≥0.6者较高,其中13~15岁人群pre-MS患病率较高。     

Follicles in pregnant rat ovary are incapable of steroidogenesis

Antral follicles are present in the ovaries throughout gestation. These follicles are “physically immature” and cannot ovulate under the induction of LH/hCG. The purpose of the present study is to examine whether follicles in pregnant rat ovaries are capable of steroidogenesis. StAR is believed to be the key regulator of steroid hormone biosynthesis. Antisense StAR probe and anti-StAR rabbit serum have been used to detect the StAR expression in ovarian follicles at various stages of pregnant rats. The results indicate that theca-interstitial cells and the membrane granulosa cells at the stages from the estrous or pre-estrous in the normal cycling rat ovary express StAR mRNA and its protein, whereas neither granulosa cells nor the theca cells in pregnant ovary throughout gestation express StAR. These results indicate that the pregnant follicles throughout gestation are incapable of steroidogenesis.     

小檗碱、隐丹参酮对胰岛素抵抗卵巢颗粒细胞凋亡相关基因的影响

通过基因芯片技术研究小檗碱、隐丹参酮对胰岛素抵抗卵巢颗粒细胞基因表达的影响。选择猪卵巢颗粒细胞为体外研究对象,利用磷酯酰肌醇-3激酶(PI-3K)特异性抑制剂——沃曼青霉素人工诱导胰岛素抵抗的细胞模型,以0.02g/L小檗碱、隐丹参酮进行干预,继续培养48h,以Trizol法提取总RNA,采用猪全基因表达谱芯片技术筛选出差异表达基因。将筛选得到差异基因输入MAS(Molecule Annotation System)中进行显著性统计分析。结果显示:小檗碱组与沃曼组比较,与细胞凋亡有关的18个基因表达发生显著变化;而隐丹参酮组有12个。这些差异表达的基因涉及TNF家族、bcl-2基因家族、Caspases家族、MAPK信号通路及凋亡传导通路、细胞循环传导通路和tgfb-smad3传导通路。通过对基因芯片上表达水平发生改变基因的分析推测,小檗碱、隐丹参酮诱导卵巢颗粒细胞凋亡是多种途径共同作用的结果,其中Fas和Caspase途径通路上的基因以及细胞周期调控相关的基因可能发挥了主要作用。     

卵巢内瘦素受体缺失对卵巢生殖功能和全身糖脂代谢的影响

为探索卵巢内瘦素信号传导缺失对卵巢自身功能和全身糖脂代谢的影响,阐明瘦素对外周生殖系统的直接作用,进行了以下研究:①对瘦素长受体缺失(LR-)的雌性B6.Cg-m / Leprdb小鼠和其同窝出生的有完整LR的雌性野生型C57BU6J小鼠(LR ),在性成熟期后的12周,进行卵巢相互移植,构建实验小鼠的LR基因型组合分别为A(躯体 ,卵巢-)、B[躯体 ,卵巢-)、C(躯体 ,卵巢 )、D(躯体-,卵巢 )、E(躯体-,卵巢-)共5组(n=5),移植后观测2个动情周期取材:②检测各自的全身糖脂代谢指标、卵巢周期和生殖激素;③卵泡刺激素(0.75 IU/g)刺激试验后,用免疫组化和蛋白质印迹(WB)方法,检测卵巢内脂代谢信号蛋白包括Janus激酶(JAK2)、磷酸化的丝裂原激活蛋白激酶(p-ERK)和低密度脂蛋白受体(LDLR)的表达.结果显示:①D、E组与A、B、C 3组比较,体重相差约1倍,性腺周围脂肪垫重量相差约10倍(P<0.01);血葡萄糖、低密度脂蛋白胆固醇(LDL-C)和胰岛素水平相差2-3倍(P<0.01);A、B、C 3组的全身血糖脂指标相似,D和E 2组的高糖脂血症升高程度也相似.(②A、B、C 3组小鼠均恢复正常的动情周期,而D、E 2组则始终处于动情间期;小鼠16周龄时D、E组与A、B、C 3组比较:卵巢重量及E2、P、FSH、LH水平显著下降(P<0.05 J.③瘦素刺激后JAK2在A、B、D和E组的卵巢中表达均呈弱阳性,而C组卵巢内呈强阳性表达;FSH刺激后p-ERK和LDLR在A、B、C 3组卵巢内均呈强阳性表达,而D和E组则表达下降.进而得出结论:①单纯卵巢内瘦素信号缺失并不影响卵巢自身的生殖功能、脂质代谢和全身的糖脂代谢.②瘦素受体全身缺失小鼠的卵巢功能障碍,与卵巢外的全身因素即高糖脂血症和低促性腺激素状态有关.③高糖脂血症降调节卵巢内的瘦素信号蛋白JAK2的表达,诱导小鼠卵巢脂质化和功能障碍;FSH刺激可诱导ERK磷酸化后LDLR的表达增强,缓解小鼠卵巢的脂质化和功能障碍.     

基因芯片研究小檗碱对胰岛素抵抗卵巢颗粒细胞基因表达的影响

应用基因芯片技术研究小檗碱对胰岛素抵抗卵巢颗粒细胞基因表达谱的影响。方法采用胰岛素信号转导通路调节糖代谢的关键分子——PI-3K的特异性阻断剂沃漫青霉素作用于猪卵巢颗粒细胞,人工诱导胰岛素抵抗(IR)的细胞模型;同时应用小檗碱作用在细胞模型上,作用48h后提取细胞总RNA,应用猪全基因组单通道芯片对样本基因进行筛查,得出差异表达基因数据并进行分析。结果显示,中药小檗碱作用模型细胞后差异表达基因42个。差异表达基因主要涉及的功能有:物质代谢、炎症与免疫反应、信号转导等。由此可得出结论:小檗碱可通过多种途径在基因水平上对胰岛素抵抗卵巢颗粒细胞起作用。     

LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid–derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.     

云南妇女卵巢癌中p21WAF基因有较高的31位密码(Ser/Arg)突变

 通过PCR-SSCP分析和序列分析,对取自云南地区的44例妇女卵巢肿瘤样本的p21^WAF基因Ser³¹密码子的突变进行了分析.结果表明35例恶性肿瘤样本中有15例发现有突变,突变率为42.9%;9例良性肿瘤中有4例出现突变,突变频率44.4%;在另外6例正常组织中出现1例突变,突变率为16.7%.这一结果表明,云南妇女在这一基因位点存在多态性,这一位点的突变具有发生卵巢癌的趋势.     

Innervation of the ovary in amphioxus: ultrastructural and immunohistochemical study

The distribution of unmyelinated nerve fiber in the ovary of amphioxus was found with transmission electron microscopic technique for the first time. The fiber is located under the ovary coat, and in close contact with it. There are two types of synaptic vesicles in the terminals of nerve fiber: one is durse-cored vesicle, the other is clear vesicle. In addition, the nerve terminals contact with follicle cells of ovary can be seen. Using immunohistochemical method. it is further demonstrated that the unmyelited nerve fiber nlay be a noradrenergic nerve fiber which is located on the ovary coat and follicle cell.     

The study of the cultivation of chinese hamster ovary and bowes cell lines with poly(organophosphazene) membranes

Sixteen poly(organophosphazenes) were prepared by reaction between polydichlorophosphazene (NPCl2)n and nucleophilic reagents such as phenoxides and amino compounds. Adhesive and colony formation percent were investigated using V-79 Chinese hamster cells on poly(organophosphazene) films. It was found that [NP(OC6H5)2-x(NHBu-n)x]n (x = 0.2, 1.8) gave the best percent adhesiveness. This value was similar to that of Falcon. On the other hand, [NP(OC6H5)-(NHBu-n)]n film showed best colony percent formation. This was of a higher value than that of Falcon. The [NP(OC6H5)2]n properties were poor for cultivation of useful Bowes and chinese hamster ovary cell lines in comparison with Cytodex III.