Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome

Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.     

多囊卵巢综合征患者卵泡基底膜超微结构的研究

运用透射电镜观察多囊卵巢综合征(PCOS)组及正常妇女组卵泡的形态,用体视学方法测量两组各级卵泡基底膜的厚度。结果显示:两组原始卵泡的基底膜厚度相似,但随着卵泡的发育卵泡基底膜逐渐增厚,PCOS组增厚更明显,对照组初级卵泡基底膜的平均厚度是0 293±0 204μm,PCOS组是0 463±0 287μm,两组比较有统计学差异(P<0 05)。对照组次级卵泡基底膜的平均厚度为0 542±0 298μm,PCOS组为1 234±0 345μm,两组比较有统计学差异(P<0 05)。两组原始卵泡的前颗粒细胞的超微结构相似,随着卵泡的发育,PCOS组的颗粒细胞内参与蛋白质合成的细胞器明显较对照组丰富;正常组窦前卵泡的颗粒细胞出现分化现象,PCOS组却未见分化现象。结果提示:PCOS患者卵泡基底膜屏障的增厚可能使卵泡刺激素FSH进入卵泡困难,颗粒细胞不能正常分化,"FSH 颗粒细胞轴"功能低下,导致不孕症。     

重组人卵泡刺激素作用下卵泡黄素化颗粒细胞M-CSF及受体表达的临床差异

目的:比较使用重组人卵泡刺激素（r-FSH）后,患者卵泡黄素化颗粒细胞巨噬细胞集落刺激因子（M-CSF）及其受体（M-CSFR）mRNA表达的差异,以探讨M-CSF及其受体对卵巢反应性的影响。方法: 接受体外受精治疗的96例多囊卵巢综合征（PCOS）患者和157例非PCOS患者,根据其使用r-FSH后反应性不同分为四组：即PCOS快、慢反应组和非PCOS快、慢反应组。收集成熟卵泡穿刺后黄素化颗粒细胞,采用SYBR Green荧光定量RT-PCR法检测颗粒细胞M-CSF、M-CSFR和管家基因GAPDH的mRNA表达,通过比较阈值法（CT值目的基因－CT值管家基因）进行基因相对定量。双独立样本t检验比较组间基因表达差异,并对差异有统计学意义的指标行Spearman相关分析检验。结果:各组患者r-FSH使用量无统计学差异（P>0.05）。PCOS患者整体与非PCOS患者整体比较,M-CSF和M-CSFR的mRNA定量均无统计学差异（P>0.05）。分组比较后,各组间M-CSF mRNA定量无统计学差异（P>0.05）。而PCOS慢反应组M-CSFR mRNA定量低于快反应组患者,差异有统计学意义（P=0.006）。对PCOS组颗粒细胞M-CSFR mRNA定量和r-FSH使用天数的Spearman相关分析提示,两者相关性有统计学意义（P=0.023）,相关系数0.511。 结论:PCOS卵巢慢反应患者对促排卵药物反应迟缓可能与其颗粒细胞M-CSFR表达减少有关。M-CSF及其受体参与影响卵巢对r-FSH的反应性。     

“Tissue Papers” from Organ‐Specific Decellularized Extracellular Matrices

Using an innovative, tissue‐independent approach to decellularized tissue processing and biomaterial fabrication, the development of a series of “tissue papers” derived from native porcine tissues/organs (heart, kidney, liver, muscle), native bovine tissue/organ (ovary and uterus), and purified bovine Achilles tendon collagen as a control from decellularized extracellular matrix particle ink suspensions cast into molds is described. Each tissue paper type has distinct microstructural characteristics as well as physical and mechanical properties, is capable of absorbing up to 300% of its own weight in liquid, and remains mechanically robust (E = 1–18 MPa) when hydrated; permitting it to be cut, rolled, folded, and sutured, as needed. In vitro characterization with human mesenchymal stem cells reveals that all tissue paper types support cell adhesion, viability, and proliferation over four weeks. Ovarian tissue papers support mouse ovarian follicle adhesion, viability, and health in vitro, as well as support, and maintain the viability and hormonal function of nonhuman primate and human follicle‐containing, live ovarian cortical tissues ex vivo for eight weeks postmortem. “Tissue papers” can be further augmented with additional synthetic and natural biomaterials, as well as integrated with recently developed, advanced 3D‐printable biomaterials, providing a versatile platform for future multi‐biomaterial construct manufacturing.     

纳米三氧化二砷磁性脂质体的热化疗作用对卵巢癌细胞nm23H1及c-myc的影响

为了研究纳米三氧化二砷磁性脂质体(NMLA)的热化疗作用对卵巢癌HO-8910细胞nm23H1和c-myc mRNA表达有无影响。以培养液做对照,分别将空白脂质体、三氧化二砷(As2O3)溶液、纳米As2O3脂质体、纳米磁性脂质体(NML)、NMLA作用于人浆液性卵巢癌HO-8910细胞,并用高频交变磁场(AMF)对经过NML和NMLA处理的HO-8910细胞作进一步热疗处理,RT-PCR方法检测各组细胞nm23H1和c-myc的mRNA表达情况。实验发现,As2O3使nm23H1mRNA的表达上调,热疗对其无影响;单纯As2O3及热疗均使c-mycmRNA的表达下调,纳米As2O3磁性脂质体的热化疗对其下调作用更为显著。上述结果表明,NMLA可在高频AMF作用下对卵巢癌细胞进行热化疗,机制与nm23H1和c-myc的表达相关。     

State of Drosophila melanogaster Ovaries after a Full Cycle of Gametogenesis under Microgravity Modeling: Cellular Respiration and the Content of Cytoskeletal Proteins

The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of Drosophila melanogaster, the full development cycle of which (from zygote to sexually mature adults) passed under simulated microgravity by a random positioning machine. The rate of cellular respiration was studied by polarography as a parameter reflecting the functional state of mitochondria. The content of cytoskeletal proteins and histones was determined using Western blotting. The relative content of mRNA was determined using qRT-PCR. The results obtained indicated an increase in the rate of cellular respiration under simulated microgravity conditions during the full cycle of gametogenesis in Drosophila melanogaster due to complex I of the respiratory chain. In addition, an increase in the contents of actin cytoskeleton components was observed against the background of an increase in the mRNA content of the cytoskeleton’s encoding genes. Moreover, we observed an increase in the relative content of histone H3 acetylated at Lys9 and Lys27, which may explain the increase in the expression of cytoskeletal genes. In conclusion, the formation of an adaptive pattern of functioning of the Drosophila melanogaster ovaries that developed under simulated microgravity includes structural and functional changes and epigenetic regulation.     

Mesenchymal Stem Cell-Conditioned Media Regulate Steroidogenesis and Inhibit Androgen Secretion in a PCOS Cell Model via BMP-2

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women. Previous studies have demonstrated the therapeutic efficacy of human bone marrow mesenchymal stem cells (BM-hMSCs) for PCOS; however, the regulatory mechanism remains unknown. Bone morphogenetic proteins (BMPs) secreted by BM-hMSCs may underlie the therapeutic effect of these cells on PCOS, based on the ability of BMPs to modulate androgen production and alter steroidogenesis pathway enzymes. In this study, we analyze the effect of BMP-2 on androgen production and steroidogenic pathway enzymes in H295R cells as a human PCOS in vitro cell model. In H295R cells, BMP-2 significantly suppressed cell proliferation, androgen production, and expression of androgen-synthesizing genes, as well as inflammatory gene expression. Furthermore, H295R cells treated with the BM-hMSCs secretome in the presence of neutralizing BMP-2 antibody or with BMP-2 gene knockdown showed augmented expression of androgen-producing genes. Taken together, these results indicate that BMP-2 is a key player mediating the favorable effects of the BM-hMSCs secretome in a human PCOS cell model. BMP-2 overexpression could increase the efficacy of BM-hMSC-based therapy, serving as a novel stem cell therapy for patients with intractable PCOS.     

表达IFNα2b-HSA融合蛋白的CHO细胞培养基优化及发酵放大

毕赤酵母表达的干扰素与白蛋白融合蛋白虽然避开了干扰素单体半衰期短的缺陷,但毕赤酵母对药物的糖基化修饰与人体的糖基化修饰差异性导致了药物的毒副作用。目前药物在CHO系统的表达得到了广泛应用。本研究室首次构建了能表达干扰素α2b和人血清白蛋白融合蛋白（IFNα2b-HSA）的CHO细胞株。在此基础上,本研究通过对12种国产商业化基础无血清培养基和5种流加培养基进行优化筛选,获得最适培养方案：基础培养基选择最适于生长的5号培养基（M2：M4=1：1）,流加培养基选择最有适于表达的F4培养基。在此基础上,进行5L生物反应器的发酵放大,pH为6.9～7.4,DO为40%～60%,细胞密度达到7.0×10⁶cells/mL时,温度由37℃降温至34℃,细胞活率降至80%时停止发酵,最终融合蛋白表达量达到137mg/L。初步实现了IFNα2b-HSA融合蛋白在CHO细胞中的高密度发酵。