健骨方对破骨细胞形成和成骨细胞增殖分化的影响

目的　探讨健骨方水提物对破骨细胞分化及成骨细胞增殖分化的影响。方法　制备健骨方水提物,通过MTT法测定药物对骨髓单核巨噬细胞（BMMs）细胞的毒性,采用核因子κB受体活化因子配体（RANKL）诱导BMMs分化形成破骨细胞,加入不同浓度药物进行干预,采用抗酒石酸酸性磷酸酶( TRACP) 染色法测定破骨细胞分化抑制作用,采用Western Blot 法测定RANKL诱导的NF-κB破骨细胞分化信号通路,运用RT-qPCR法测定信号通路下游破骨细胞分化关键基因NFATc1、C-FOS等的mRNA表达水平。以MC3T3-E1细胞作为前体成骨细胞,加入不同浓度药物进行干预,通过CCK8法测定细胞增殖能力、PNPP法检测碱性磷酸酶（ALP）活性、茜素红S染色法测定细胞矿化能力。结果　MTT法结果显示,健骨方细胞有毒性浓度大于500 μg/mL（P＜0.05）,破骨细胞分化抑制IC50为1.25 μg/mL。机制研究显示健骨方显著下调了RANKL-NF-κB信号通路中的p-P65、P53的蛋白表达（P＜0.05）,显著抑制了通路下游C-FOS、NFATc1等的mRNA表达水平（P＜0.01,P＜0.05）。此外,成骨细胞活性检测显示,健骨方能明显促进MC3T3-E1细胞增殖、提高ALP活性及增加成骨细胞钙化的能力。结论 健骨方具有抑制破骨细胞分化和促进成骨前体细胞增殖、分化、矿化的药效作用。其作用与抑制破骨细胞分化RANKL-NF-κB信号通路及其下游C-FOS、NFATc1等基因,上调成骨细胞分化促进因子CAL1A2、SPARC和FOSL1基因的表达有关。     

人骨形成蛋白2的基因克隆和序列分析

目的　探讨克隆人骨形成蛋白 2基因编码区的方法。方法　由人末梢血白细胞中提取细胞总DNA ,利用PCR法 ,从DNA中分别扩增出人骨形成蛋白 2外显子编码区基因DNA片段 ;将获得的基因片段插入PGEM -T -Easy质粒中 ,转化到大肠杆菌DH5α后挑选阳性克隆 ,利用限制性内切酶酶切核苷酸序列分析鉴定后 ,连接获得人骨形成蛋白 2基因。结果　通过质粒DNA酶切分析及序列测定 ,我们获得的基因片段为人骨形成蛋白 - 2DNA序列。结论　首次由人白细胞DNA中克隆获得人骨形成蛋白 2全长基因 ,为表达、应用骨形成蛋白 2提供了前提条件     

TZDs对骨髓破骨细胞生成作用和 成骨细胞生成作用的影响

[目的]体外观察TZDs类药物对小鼠原代骨髓细胞向成骨细胞和破骨细胞分化的影响,探讨其对糖尿病患者骨骼代谢的影响和作用机制.[方法]无菌条件下从小鼠股骨分离获取骨髓细胞,贴壁细胞进行成骨细胞生成实验研究,悬浮细胞进行破骨细胞生成实验研究.随后在1、2、5μmol/L罗格列酮干预下诱导成骨细胞分化培养3周,进行VonKossa染色观察成骨细胞矿化结节,并测定成骨细胞标记物ALP活性、BMP-2及TGF-β分泌的影响;观察罗格列酮对破骨细胞形成和成熟的影响及标志性基因表达.[结果]1、2、5μmol/L罗格列酮干预组与对照组相比,成骨细胞的钙结节形成比例显著降低(P＜0.05),ALP、BMP-2、TGF-β水平呈剂量依赖性地下降(均P＜0.05);同时剂量依赖性促进c-fos和RANK基因的表达,破骨细胞生成实验显示罗格列酮促进破骨细胞分化发育和成熟.[结论]罗格列酮可剂量依赖性地抑制骨髓细胞向成骨细胞分化,促进向破骨细胞分化.有助于理解Ⅱ型糖尿病患者在应用TZDs骨丢失的原因.     

Ⅱ型单纯疱疹病毒培养及gG-2基因特异片段的克隆

目的:通过培养和扩增Ⅱ型单纯疱疹病毒(HSV-2)并提取其全基因组,从而克隆包膜糖蛋白gG-2全长基因及其保守性较高、抗原性较强的特异片段。方法:将HSV-2病毒感染Hela细胞获得大量病毒液,用酚-氯仿法提取病毒全长基因组,并以此为模板扩增编码gG-2的US4基因,再根据氨基酸序列比对结果,选取其特异片段,设计带酶切位点的引物克隆gG-2基因特异片段。结果:成功地获得了大量HSV-2病毒液并提取了病毒基因组,克隆了gG-2全长基因以及特异片段,测序证实,两者均与GenBank中报道的序列相一致。结论:对于遗传特性相对稳定的HSV-2来说,通过病毒感染细胞的方法短时期内获得大量病毒液,并从病毒全基因组中克隆gG-2特异基因片段的方法是可行的,从而为进一步构建gG-2特异片段相关载体、表达相应蛋白奠定了基础。     

人骨形成蛋白2的基因克隆和序列分析

目的 探讨克隆人骨形成蛋白2基因编码区的方法。方法 由人末梢血白细胞中提取细胞总DNA，利用PCR法，从DNA中分别扩增出人骨形成蛋白2外显子编码区基因DNA片段；将获得的基因片段插入PGEM—T—Easy质粒中，转化到大肠杆菌DH5α后挑选阳性克隆，利用限制性内切酶酶切核苷酸序列分析鉴定后，连接获得人骨形成蛋白2基因。结果 通过质粒DNA酶切分析及序列测定，我们获得的基因片段为人骨形成蛋白—2DNA序列。结论 首次由人白细胞DNA中克隆获得人骨形成蛋白2全长基因，为表达、应用骨形成蛋白2提供了前提条件。     

JNK抑制剂SP600125对骨质疏松症保护作用的实验研究

目的 探讨JNK抑制剂SP600125对骨质疏松症模型成骨细胞和破骨细胞分化及功能的影响。方法 使用CCK8试验检测RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性;使用相关染色试验探究JNK抑制剂SP600125的成骨化作用和对破骨细胞分化及功能的影响;使用RT-PCR分别检测BMMs细胞中破骨细胞及成骨细胞特异性基因mRNA表达水平;使用蛋白印迹试验检测RAW264.7细胞中JNK通路和NF-κB通路的活化水平;使用DCFH-DA法检测RAW264.7细胞中ROS水平。结果 CCK8试验结果显示,当SP600125浓度≤20 μmmol/L,对RAW264.7细胞、BMMs细胞和成骨细胞的增殖活性无显著影响;SP600125不影响成骨细胞钙结节的形成并抑制破骨细胞分化和功能;SP600125抑制的破骨细胞特异性基因的表达但不改变成骨细胞特异性基因的表达;SP600125抑制RANKL诱导的RAW264.7细胞中JNK通路和NF-κB通路的激活及ROS水平的升高。结论 JNK抑制剂SP600125能够抑制破骨细胞的分化及骨吸收功能,但对成骨细胞的分化无显著影响。     

人骨形成蛋白1全基因cDNA的克隆与序列分析

目的　克隆人骨形成蛋白１（ Ｏ Ｐ１） 全基因ｃ Ｄ Ｎ Ａ,研究其结构与功能。方法　取新鲜胎盘组织提取总 Ｒ Ｎ Ａ,分别以 Ｏｌｉｇｏ（ｄ Ｔ） 、随机引物及特异引物 Ｒ Ｔ Ｐ Ｃ Ｒ。以 Ｓａｎｇｅｒ 双脱氧链终止法作序列测定。结果　以 Ｏｌｉｇｏ（ｄ Ｔ） 引导的 Ｒ Ｔ Ｐ Ｃ Ｒ 仅扩增出 Ｏ Ｐ１ 基因３′端５６０ｂｐ 片段,以随机引物及特异引物引导的 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增出５′端７５０ｂｐ 片段,重组连接两片段得到 Ｏ Ｐ１ 全基因ｃ Ｄ Ｎ Ａ;序列测定显示５ 个核苷酸变异,但编码的氨基酸相同。结论　首次从中国人胎盘组织克隆到 Ｏ Ｐ１ 全基因ｃ Ｄ Ｎ Ａ。与 Ｇｅｎ Ｂａｎｋ 中的 Ｏ Ｐ１ 序列比较,中国人 Ｏ Ｐ１ 基因之中存在５ 个简并密码子。     

破骨细胞分化因子胞外区的克隆表达与纯化

目的　破骨细胞分化因子的克隆 ,重组蛋白的表达及纯化。方法　自 1g死胎肝组织中提取总RNA ,反转录cDNA后作为模板 ,设计上下游引物 ,多聚酶链反应 (PCR)扩增出目的片段。将PCR产物克隆至组氨酸标签的融合蛋白表达载体 pProEXTM HTc ,并送测序。在异丙基硫代 β D 半乳糖苷 (IPTG)诱导下 ,可以稳定表达出破骨细胞分化因子胞外区蛋白 ,用镍离子交换柱纯化该蛋白。结果　逆转录 多聚酶链反应 (RT PCR )扩增出特异的目的条带 ,75 0bp大小。测序结果完全正确。诱导后可以表达重组蛋白 ,相对分子质量为 3 2× 10 3 。结论　RT PCR可以简便的扩增目的基因。重组破骨细胞分化因子蛋白可以通过大肠杆菌原核表达载体诱导表达得到。     

中国人血管生成素-1cDNA克隆和序列分析

目的 克隆和测定国人血管生成素-1(Ang-1)基因序列。方法 巢式多聚酶链反应(PCR)从婴儿肺组织扩增中国人Ang-1cDNA，克隆入载体pGEM-Teasy，测定核苷酸序列，分析测序结果。结果 通过获得Ang-1cDNA，和GeneBank的Ang-1序列(U8508)对照，两者成熟区cDNA的核苷酸和氨基酸同源性分别大于99％和大于98％；在330bp～930bp问核苷酸同源性为98％，氨基酸同源性为95％。结论 获得克隆的中国人Ang-1基因成熟区。其卷曲卷曲结构域内可能存在一个内部高变区。     

钝齿棒杆菌产精氨酸代谢途径中argB基因的扩增及其序列分析

根据GenBank报道的谷氨酸棒杆菌ATCC13032序列,设计并合成一对引物,获得了全长argB基因片段.将其克隆到pGEM T Easy载体中,经测序鉴定后,以其为DIG标记探针,通过SouthernBlot分析钝齿棒杆菌A.S1.542与谷氨酸棒杆菌ATCC13032,argB基因核苷酸同源性高达99%,氨基酸序列95%相同.     

Livin基因siRNA重组表达载体的构建

目的构建针对人抑凋亡基因Livin的小干扰RNA（siRNA）重组表达质粒载体。方法通过分子克隆技术,设计并合成具有基因特异性的3组寡核苷酸片段,克隆到pmU6质粒载体,并经菌落PCR鉴定及测序鉴定,Livin的siRNA序列成功克隆到重组表达质粒载体中。结果经菌落PCR鉴定及测序鉴定证实,人Livin的siRNA序列成功克隆到表达载体中,插入序列正确。结论成功构建了Livin siRNA质粒表达载体,为后续研究降低或沉默Livin基因表达后能否有效抑制膀胱癌细胞增殖和促进细胞凋亡奠定了基础。     

Isolation and characterization of a novel cDNA encoding a DNA-binding protein (Hils1) specifically expressed in testicular haploid germ cells

A cDNA encoding a protein homologous with histone H1 has been cloned from a haploid germ cell specific cDNA library. Deduced amino acid sequence (170 amino acids) showed 40% identity with histone H1 globular domain. Messenger RNA of the gene was observed exclusively in the testis, and was accumulated after post-natal day 23. Western blotting analysis showed that the protein encoded by this gene is about 19 kDa in molecular weight, and it was exclusively recovered from the nuclei of testicular germ cells. Immunohistochemical analysis showed that the protein was localized to the nuclei of round and elongating spermatids, consistent with the results of immunoblot analysis. Thus, the gene product was named Hils1 (histone H1 like protein in spermatids 1). In vitro DNA-binding experiments using DNA-cellulose mini-columns showed that Hils1 was able to bind to both double and single stranded-DNAs in a non-sequence-specific manner. These findings suggest that Hils1 may play an important role in the structural changes of spermatid nuclei, such as nuclear condensation, and gene regulation of haploid germ cell differentiation.     

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.     

人白细胞介素-10基因的克隆与逆转录病毒载体的构建

目的克隆、测序人白细胞介素(hIL)-10基因片段,构建逆转录病毒载体(MSCV- hIL-10)。方法采用刀豆蛋白A活化的人外周血单个核细胞培养提取总RNA,设计随机引物并逆转录反应合成hIL-10 cDNA第1链,并以hIL-10特异性引物行PCR扩增,获得hIL-10克隆基因片段,将hIL-10克隆基因片段,经双酶切后定向插入到逆转录病毒载体(MSCV)中,用脂质体法转染PT67包装细胞,以G418筛选阳性克隆。结果聚合酶链反应(PCR)扩增出534 bp特异性片段,测序结果表明同源性与GenBank报道完全一致,hIL-10基因片段插入MSCV载体中经转染包装后得到2×10~7 CFU／ml的分泌hIL-10的病毒悬液,hIL-10的分泌量为1220ng／10~6细胞·d~(-1)。结论成功克隆hIL-10开放阅读框架,成功构建MSCV-hIL-10重组质粒,获得高滴度的分泌hIL- 10的病毒悬液,hIL-10的表达可以应用于移植排斥的基因治疗。     

Chicken parathyroid hormone-related protein and its expression during embryologic development.

A parathyroid hormone-related protein (PTHrP) is the probable cause of humoral hypercalcemia in malignancy, but its normal physiologic role remains unknown. Since current evidence suggests that PTHrP may have a role in embryonic development, we cloned a genomic fragment that encodes chicken PTHrP (cPTHrP) and studied the expression of PTHrP in developing chick embryos. Blot hybridization of chicken genomic DNA with a cPTHrP genomic DNA probe showed a single band, suggesting that a single-copy gene encodes cPTHrP. By screening a chicken genomic library with the human probe an open reading frame was identified that corresponds to the human PTHrP (hPTHrP) exon IV. Compared to the human sequence the 5' splice junction is highly conserved and the two predicted propeptide residues are identical. The sequence predicts a mature peptide of 139 amino acids; all of the first 21 and 94 of the first 112, but only 8 of the final 27 residues of cPTHrP are identical to the human sequence. The structural features required for PTH receptor binding and activation are highly conserved between chicken and hPTHrP. Poly(A)-enriched RNA from 3-15 day chicken embryos was surveyed by hybridization to the chicken probe. A hybridizing band of 1.45 kb was found in tissues derived from all three germ layers, including brain, heart, lung, liver, gizzard, intestine, chorioallantoic membrane, yolk sac, and skeletal muscle. An additional 1.2 kb hybridizing band was found in some tissues. The conservation of the PTHrP sequence between chicken and mammals supports the view that PTHrP has an important physiologic role.(ABSTRACT TRUNCATED AT 250 WORDS)     

人精子表面蛋白P34H的基因克隆及其在睾丸和附睾中表达分析

目的 :克隆精子表面蛋白P34H基因的编码区 ,为进一步体外表达P34H蛋白做准备。　方法 :提取人附睾体部总RNA ,并以此为模板 ,进行反转录PCR获得编码P34H蛋白的基因片段。应用T/A克隆策略 ,将扩增的P34H基因编码区克隆入T载体 ,并通过双酶切和DNA测序进行鉴定。同时 ,以 β actin为内参照物 ,进行反转录PCR半定量分析 ,比较P34H在附睾头部、体部和尾部及睾丸组织中的表达量。　结果 :成功地克隆了P34H基因。将P34H的cDNA序列登录GenBank ,登录号为AF5 15 6 2 5。反转录PCR半定量分析表明P34H主要在附睾体部表达。　结论 :克隆的P34H基因可用于构建表达载体     

Molecular Cloning of Human Sperm Surface Protein P34H Gene and Semi-quantitative Analysis of Its Expression in Testis and Epididymidis

Molecular Cloning of Human Sperm Surface Protein P34H Gene and Semi-quantitative Analysis of Its Expression in Testis and Epididymidis