重组人组织激肽释放酶基因转染人脐静脉内皮细胞

目的　探讨重组人组织型激肽释放酶 (HK)基因腺相关病毒载体 (rAVV/HK)对体外培养的人脐静脉内皮细胞 (HUVECs)作用。方法　将HK基因定向克隆入腺相关病毒质粒 pAAV MCS中 ,形成重组腺相关病毒 (rAAV HK)质粒 ,经聚合酶链反应 (PCR)检测和DNA测序后 ,将rAAV HK、pAAV Helper和 pAAV RC 3种质粒通过脂质体共转染 2 93细胞 ,包装成带有HK目的基因的重组rAVV/HK ,采用 β半乳糖苷酶原位染色法测定报告基因rAVV/LacZ在HT10 80中的表达 ,间接计算病毒滴度 ,并观察转染基因在传代细胞中的表达。在相同的条件下体外分别培养HUVECs(对照组 )和rAVV/HK转染的HUVEC(HK转染组 ) 4 8h ,采用逆转录聚合酶链反应 (RT PCR)技术检测两组HUVECsHKmRAN的表达 ,并利用硝酸还原酶法和分光光度法分别检测两组HUVECs一氧化氮(NO)含量和一氧化氮合酶 (NOS)活性。结果　rAVV/HK病毒滴度为 6 .2× 10 7个 /mL ,LacZ基因能够在第一传代和第六代HT10 80细胞内稳定持续表达。与对照组比 ,HK转染组HUVECsHKmRNA表达增加 (P <0 0 0 1) ;而且HK转染组HUVECs细胞培养液中NO的含量和NOS活性明显增高 ,P值分别 <0 0 1和 <0 0 5。结论　HK基因通过重组腺相关病毒载体可成功转染体外培养的HU VECs ,使其HKmRNA表达增加 ,NOS活性和NO含量升     

人激肽释放酶重组腺相关病毒对人脐静脉内皮细胞的影响

目的:构建携带人激肽释放酶(kallikrein,KK)基因的重组腺相关病毒载体,并导入体外培养的人脐静脉内皮细胞(HUVEC),观察其对缓激肽(BK)、一氧化氮(NO)、内皮素-1(ET-1)水平和内皮型一氧化氮合酶(eNOS)基因表达的影响。方法:(1)将KK基因定向克隆入腺相关病毒载体质粒pAAV-MCS中,构建成pAAV-KK;(2)分别将pAAV-KK及pAAV-LacZ与另外两种质粒pHelper、pAAV-RC通过脂质体共同转染AAV-293细胞,包装出rAAV-KK和rAAV-LacZ重组腺相关病毒。将rAAV-LacZ原液稀释成不同浓度梯度并感染HT-1080细胞,通过β-半乳糖苷酶染色测定病毒感染滴度,并观察LacZ传代HT-1080细胞的表达情况;(3)将rAAV-KK感染HUVEC,检测其对缓激肽、NO、ET-1水平和eNOS基因表达的影响。结果:(1)成功构建了带有KK目的基因的重组腺相关病毒载体和带有LacZ报告基因的重组腺相关病毒载体(对照载体);(2)rAAV-LacZ重组病毒以感染滴度6.2×107I U/ml感染HT-1080细胞后,LacZ基因能够在连续传代的HT-1080细胞内稳定持续表达;(3)与空白组、LacZ组(对照组)相比,KK感染组的HUVEC细胞培养液中BK[(2.864±1.36)pmol/ml∶(2.782±1.48)pmol/ml∶(6.576±2.08)pmol/ml]、NO[(16.42±1.02)μmol/L∶(16.46±1.25)μmol/L∶(21.28±1.46)μmol/L]水平明显增加,eNOS基因[(0.353±0.031)∶(0.364±0.049)∶(0.423±0.038)]表达明显增强,ET-1[(262.91±17.85)pg/ml∶(263.56±15.87)pg/ml∶(199.48±16.99)pg/ml]水平显著下降(P均0.01)。结论:(1)成功构建了携带有人激肽释放酶基因的重组腺相关病毒,rAAV-KK感染体外培养的人脐静脉内皮细胞;(2)可使细胞分泌BK、NO,以及表达eNOS基因增加,ET-1减少,改善内皮细胞功能。     

人组织激肽释放酶基因治疗对2型糖尿病大鼠胰岛素抵抗和肾脏并发症的影响

目的 探讨人组织激肽释放酶(HK)基因对2型糖尿病大鼠胰岛素抵抗和慢性肾脏并发症的治疗作用及其机制.方法 在雄性Wistar大鼠以高脂高糖饲料加小剂量链脲佐菌素建立2型糖尿病动物模型.以重组腺相关病毒为载体介导HK基因(HK组)或对照基因LacZ(LacZ组)在糖尿病大鼠体内表达,观察实验动物血压、血糖、血胰岛素、尿微量白蛋白、尿渗透压的变化.计算稳态模型评估的胰岛素抵抗指数(HOMA-IR)、内生肌酐清除率(Ccr)和尿白蛋白排泄率(UAER).Western印迹法检测肝脏、骨骼肌磷脂酰肌醇3激酶(P13K)的110亚基(pll0)和蛋白激酶B(Akt/PKB)第308位苏氨酸(pThr 308)的磷酸化水平.肾脏切片作形态学分析.结果 HK组第2周开始出现血压下降,并一直持续到实验结束(12周时),而LacZ组血压无明显下降.HK组和LacZ组大鼠空腹血糖无明显变化[(13.09±3.01 vs 13058±2.88)mmol/L],但两组空腹血胰岛素水平[(8.19±2.45 vs 13.85±3.76)mIU/L,P<0.01]和HOMA-IR(4.76±0.33 vs 8.36±0.48,P<0.01)差异有统计学意义.HK组大鼠肌肉和肝脏pll0和pThr308的表达明显增加.HK组UAER[(7.90±2.76 vs 19.07±9.17)mg/24h,P<0.01]和Ccr(0.16±0.12 vs 0.43±0.25,P<0.01)明显低于LacZ组,而尿渗透压明显高于LacZ组[(1150.3±301.9 vs 737.8±184.5)mmol/L,P<0.05].肾脏病理学结果显示LacZ组肾小球和肾小管损害明显,而HK组明显减轻.结论 重组腺相关病毒介导HK基因表达可能通过增加PI3K/Akt磷酸化明显改善2型糖尿病大鼠胰岛素抵抗,同时明显减轻糖尿病肾脏损害.     

激肽释放酶腺相关病毒载体的构建及对脐静脉内皮细胞功能的影响

目的构建激肽释放酶(HK)腺相关病毒(AAV)载体,检测重组病毒感染脐静脉内皮细胞系(HUVEC)后HK基因和内皮功能相关基因表达的变化。方法HK基因克隆人腺相关病毒载体质粒pAAV-MCS,和腺相关病毒包装质粒pAAV-RC、腺病毒辅助质粒pHe1per共转染293细胞,包装成带有HK基因的重组腺相关病毒(rAAV-HK)。以rAAV-HK感染HUVEC。RT-PCR法检测HK基因、内皮型一氧化氮合酶(eNOS)、凋亡蛋白酶(caspase-3)、内皮素-1(ET-1)、血管内皮生长因子(VEGF)、内皮素B_1受体以及缓激肽B_1受体、缓激肽B_2受体的mRNA转录变化。ELISA法测定HUVEC上清液和胞内HK的含量。结果成功构建了rAAV-HK。rAAV-HK感染HUVEC后,实验组胞内HKmRNA转录(0．59±0．12)比空白组(0．26±0．05)明显增加(P＜0．01)；实验组胞内HK含量(120．1±40．9)比空白组(30．8±12．8)显著升高(P＜0．01)；实验组eNOSmRNA转录(1．19±0．28)较空白组(0．66±0．11)增加(P＜0．05),实验组caspase-3mRNA转录(0．30±0．25)较空白组(0．67±0．27)减弱(P＜0．05)。VEGF、内皮素-1、内皮素B_1受体、缓激肽B_1受体、缓激肽B_2受体mRNA转录两组差异无统计学意义。结论rAAV-HK病毒感染HUVEC后能高效表达HK,并促进HUVEC胞内eNOSmRNA转录,抑制caspase-3mRNA转录,提示导入HK基因能够改善内皮功能。     

人激肽释放酶基因对肾性高血压大鼠主动脉胶原含量的影响及其机制探讨

目的 观察重组腺相关病毒介导人激肽释放酶基因对肾性高血压大鼠主动脉胶原含量的影响及其可能的机制. 方法 将人组织型激肽释放酶基因(HK)克隆入重组腺相关病毒载体(rAAV)中,经三质粒共转染方法在HEK293细胞中包装成rAAV-HK病毒.雄性Wistar大鼠24只,随机分为假手术和模型组,采用5/6肾切除的方法构建肾性高血压大鼠模型.模型组大鼠再随机分成单纯手术(n=6)、对照组(n=6)和实验组(n=6).实验组大鼠单次尾静脉注射1×1011pfU的rAAV-HK病毒.分别于模型构建前、后(基因转染前)及基因转染后1～3月测大鼠尾动脉血压.基因转染3月后处死动物,取其心脏、主动脉和肾脏等脏器提取总RNA及蛋白质.主动脉组织切片行天狼星红染色,Western Blot检测主动脉组织缓激肽B2和血管紧张素Ⅱ1型受体(AT1R)蛋白表达水平. 结果 HK基因转染后3月,实验组大鼠与单纯手术和对照组相比血压明显下降[(163±13)vs(217±16)vs(220±13)mmHg,n=6,P＜0.01];主动脉中膜内胶原纤维明显减少;主动脉组织缓激肽B2受体蛋白表达水平明显上调;AT1R蛋白表达水平则明显下调. 结论 重组腺相关病毒介导人激肽释放酶基因能够明显降低肾性高血压大鼠血压,同时减少主动脉中膜胶原纤维含量,其机制可能与它上调缓激肽B2受体和下调AT1R蛋白在主动脉组织的表达水平有关.     

人激肽释放酶腺相关病毒载体的构建及其在人脐静脉内皮细胞中的表达

目的研究人激肽释放酶腺相关病毒载体的构建,观察重组病毒感染人脐静脉内皮细胞株后人激肽释放酶基因的表达。方法将人激肽释放酶基因定向克隆入AAV载体质粒pAAV-MCS中,并与两种辅助质粒pAAV-RC和pHelper共转染293细胞,包装成带有人激肽释放酶基因的重组腺相关病毒(rAAV);收集病毒颗粒并测定病毒滴度。以不同滴度的病毒分别感染人脐静脉内皮细胞,逆转录聚合酶链反应定量检测人激肽释放酶在该细胞中的表达。酶联免疫吸附法测定人脐静脉内皮细胞内人激肽释放酶的含量。结果成功获得了重组人激肽释放酶基因AAV载体,重组病毒滴度为6.2×1010个/L。以滴度分别为1×109个/L、1×108个/L和1×107个/L的病毒感染人脐静脉内皮细胞,与空白对照组比较,人激肽释放酶的表达均有增加(P<0.05),但以1×109个/L组升高最明显(P<0.001)。结论带有人激肽释放酶的重组腺相关病毒滴度可以稳定地达到1010个/L以上,感染人脐静脉内皮细胞后,人激肽释放酶基因在宿主细胞中的表达明显增强。     

重组腺病毒介导的人一氧化氮合酶基因转染对大鼠颈总动脉球囊损伤后新生内膜的影响

目的 :研究重组腺病毒介导的人内皮型一氧化氮合酶基因 (eNOS)表达生成的一氧化氮合酶 (NOS)对球囊损伤后大鼠颈总动脉新生内膜的抑制作用。方法 :在 2 93细胞内扩增、纯化Ad LacZ和Ad eNOS ,鉴定其是否携带有LacZ和eNOS基因。建立大鼠颈总动脉球囊损伤模型后 ,将磷酸缓冲液 (PBS)、Ad LacZ和Ad eNOS在体内分别转染到损伤血管段 ,以X gal染色、苏木精 伊红染色 ,免疫组化及计算机图像分析处理等方法观察转染动脉节段外源性eNOS蛋白表达及其对新生内膜的影响。结果 :重组腺病毒携带有eNOS基因 ,并且在损伤血管段得到有效表达。转染后PBS组、Ad LacZ组和Ad eNOS组的新生内膜面积分别为 (0 .187± 0 .0 18)、(0 .134± 0 .0 6 1)和 (0 .0 6 3± 0 .0 2 6 )mm 2 ,新生内膜与中膜面积比值 (I/M)分别为 1.5 76± 0 .2 73、1.342± 0 .35 7和 0 .5 6 0± 0 .16 1。与PBS组、Ad LacZ组相比Ad eNOS组无论新内膜面积 ,还是管腔狭窄程度都明显减小。结论 :腺病毒介导的eNOS基因转染能有效抑制球囊损伤后血管内膜的增生 ,可防治血管成形术后再狭窄     

人组织因子途径抑制物腺病毒表达系统的构建及体内表达

目的构建携载人组织因子途径抑制物(tissuefactorpathwayinhibitor,TFPI)基因的重组腺病毒载体,为基因治疗提供实验基础。方法利用基因重组技术,将人TFPI基因连接到穿梭质粒pDC316中,然后将腺病毒骨架质粒pBHGlox△E1,3Cre以及重组穿梭质粒pDC316-TFPI共转染293细胞,并在其中发生Cre重组酶介导的位点,特异性重组及腺病毒包装,扩增后进行滴度测定。将包装成功的携带人TFPI基因的重组腺病毒(Ad-TFPI)转染兔颈动脉,并用携带LacZ报告基因的重组腺病毒(Ad-LacZ)作为对照,3d后RT-PCR、ELISA法检测人TFPImRNA、蛋白的表达。结果得到了携带人TFPI基因的重组腺病毒,包装的病毒蚀斑形成单位(plaqueformationunit,PFU)滴度为7.6×1012/L。在Ad-TFPI转染兔颈动脉后3d,RT-PCR法和ELISA法均检测出TFPI表达,Ad-LacZ转染后未测到人TFPI的表达。结论成功构建了人TFPI腺病毒表达载体,为下一步的基因治疗提供了基础。     

腺相关病毒介导的仓鼠δ-SG基因在TO-2型仓鼠骨髓间充干细胞的表达

目的构建含有δ-SG基因的腺相关病毒载体并检测经腺相关病毒介导基因转染的TO-2型仓鼠骨髓间充质干细胞(MSCs)中目的基因的表达。方法用EcoRI酶和XhoI酶酶切含有δ-SG基因的pUC57-SG质粒,获得δ-SG基因。片段回收后定向插入腺相关病毒载体质粒pITR-GFP中重组成pITR-GFP-SG,并同pXX680、pHelper三质粒经钙磷沉淀法共转染HEK293细胞,斑点杂交法测定重组病毒颗粒滴度,再将重组病毒颗粒转染TO-2型仓鼠的MSCs,利用RT-PCR法,免疫荧光检测目的基因的表达,MTT检测转染细胞增殖能力。结果成功构建了δ-SG基因的腺相关病毒载体。含δ-SG基因的腺相关病毒感染TO-2型仓鼠MSCs48h后,RT-PCR产物经琼脂糖凝胶电泳出现884bp-DNA片段;免疫荧光检测显示TO-2型仓鼠MSCs中呈现绿色颗粒,大小深浅不一;MTT证实转染后细胞的增殖能力未受明显影响。结论采用腺相关病毒介导的方法,可以将δ-SG基因导入TO-2型仓鼠的MSCs并成功表达。     

Human retroviruses

After many unsuccessful years of searching, the first pathogenic human retrovirus, the human T-cell leukaemia lymphoma virus (HTLV-I), was reported as recently as 1980 and since that time has been causally linked to the adult T-cell leukaemia lymphoma syndrome. A second HTLV (HTLV-II) isolated shortly afterwards is less clearly linked to some leukaemic and chronic lymphoid malignancies. The second major family of human retroviruses are the human immunodeficiency viruses (HIV) the first group of isolates (HIV-I) of which cause the acquired deficiency syndrome (AIDS). A second group of these viruses (HIV-II), have recently been identified in West Africa. They appear to be less clearly associated with disease and more similar in molecular structure to the Simian immunodeficiency viruses. AIDS has now become a major global pandemic, and vaccine and therapeutic strategies are urgently being investigated in an effort to control the disease. Unfortunately, current results are not very encouraging. In the meantime, preventative and educational measures are of utmost priority in order to prevent further spread. It is not unlikely that new human retroviruses will be discovered over the next few years.     

Human parvoviruses

Human parvovirus B19 is a common infection that causes a number of clinical illnesses, most of which are benign. However, in patients with a need for increased red cell production, a deficient immune system, or both (such as occurs in the fetus), it can be a life-threatening infection. Intravenous immunoglobulin should be considered in the treatment of chronic B19 infection in immunodeficient patients and possibly in the management of other serious B19 infections. Recent advances in the laboratory, especially the increased sensitivity of polymerase chain reaction assay for detecting B19 DNA and the development of a cell line that produces B19 proteins in the form of empty capsids, should lead to significant advances in our understanding, treatment, and management of B19 disease in the near future.     

Human microsporidioses

Recent developments in microsporidiosis research include the increased utilization of molecular techniques for the investigation of clinical specimens as well as for epidemiological and phylogenetic studies. Special attention is given to studies reporting severe disseminated microsporidioses involving most organs in AIDS patients, and the increased number of HIV-seronegative and immunocompetent individuals with microsporidiosis. The potential efficacy of fumagillin (Sanofi Recherche, Gentilly, France) in infections caused by Enterocytozoon bieneusi, and the remission of intestinal microsporidiosis in HIV infected patients with antiretroviral therapy are also highlighted.     

Human rabies

A 40-year-old man who farmed in Mexico and raised dogs as a hobby presented with dysphagia, hydrophobia, insomnia, anorexia, malaise, fever, and decreased strength and sensation in his dominant arm. After a repetitive three-hour history and physical examination, a tentative diagnosis of rabies was made in an atmosphere of patient denial followed by reluctance of hospital personnel to accept such a rare diagnosis. Upon confirmation of the diagnosis by the Center for Disease Control, Atlanta, the patient underwent aggressive therapy, including maximum respiratory support, anticonvulsants, steroids, pressors, hemodialysis and interferon treatment, but died on the 16th day following admission. This case is presented because of its rarity and to review the disease, clinical history, current therapy, and recent literature regarding emergency department differential diagnosis.     

Human babesiosis

Human babesiosis is an emerging intraerythrocytic infection caused by protozoal parasites transmitted by ixodid ticks. Babesiosis is endemic in the northeastern and upper midwestern regions of the United States and is found sporadically in other parts of the United States, Europe, Asia, Africa, and South America. Babesial infections range from asymptomatic to severe and occasionally are fatal. Specific laboratory diagnosis of babesial infection is made by morphologic examination of Giemsa-stained blood smears, serology, and amplification of babesial DNA using polymerase chain reaction. The combination of atovaquone and azithromycin is the treatment of choice for mild-to-moderate illness, whereas clindamycin and quinine and exchange transfusion are indicated for severe disease.     

Human prorenin

Human prorenin is the enzymatically inactive biosynthetic precursor of renin. Recent interest has focused on the posttranslational sorting and processing of prorenin to renin since markedly increased levels of circulating prorenin have been associated with both physiological and pathological changes. These observations raise the question of whether prorenin processing may be a regulatory event in renin production in the kidney. In the juxtaglomerular cells of the kidney, prorenin can be sorted to either of two pathways: 1) the regulated pathway, which is mediated by secretory granules, where a thiol protease resembling cathepsin B processes prorenin to renin by cleavage of the amino terminal 43-amino acid prosegment, which allows exposure of the active site of renin, or 2) the constitutive pathway, which is not regulated and does not involve conversion of prorenin to renin. Studies in which segments of prorenin are modified by site-directed mutagenesis suggest that the prosegment and glycosylation are not required for sorting, although they may influence or participate in sorting, or both. Certain areas in the prosegment are important determinants of enzyme activity and ability to cleave the prosegment. Further structural analysis of prorenin will be useful to assess details of its sorting and processing. In addition, a number of extrarenal tissues such as uterine lining, ovarian theca, corpus luteum, pituitary, and adrenal, express the renin gene. These tissues have different capabilities to sort and process prorenin compared with kidney, and some tissues secrete only prorenin. Whether prorenin-to-renin conversion is necessary to activate these local renin-angiotensin systems is a key issue.(ABSTRACT TRUNCATED AT 250 WORDS)