绿色荧光蛋白基因在骨髓基质细胞内表达的意义

在体外分离和扩增大鼠骨髓基质细胞,用脂质体转染法介导绿色荧光蛋白基因进入骨髓基质细胞内,荧光显微镜下检测荧光蛋白的表达,台盼蓝排斥试验检测转染细胞的活力。结果绿色荧光蛋白在转染大鼠骨髓基质细胞24h后开始表达,48~96h表达最强,转染效率为(28.9±3.6)%;然后逐渐降低,2周后仍可见少量表达。提示外源基因可以在骨髓基质细胞内高效表达。     

绿色荧光蛋白基因转染骨髓间质干细胞

目的 建立以增强型绿色荧光蛋白基因示踪骨髓间质干细胞的方法。方法 采用密度梯度离心法分离、培养免骨髓间质干细胞，以脂质体介导法转染增强型绿色荧光蛋白基因的表达质粒，荧光显微镜观察基因表达及转染效率。结果 绿色荧光蛋白在基因转染12h后开始表达，48～72h达高峰，并在1周内有较强的表达，以后逐渐减弱，4周左右仍有少量表达，转染效率与质粒、脂质体的浓度有关，外缘基因导入后不影响骨髓间质干细胞的生长和增殖。结论 脂质体介导的绿色荧光蛋白基因转染骨髓间质干细胞后能安全、有效地表达，转染效率为20％～30％，是一种较为理想的干细胞示踪方法。     

绿色荧光蛋白/PAP-a融合表达载体的构建及其在SMMG-7721细胞中的表达

目的构建绿色荧光蛋白/PAPa融合表达载体,并检则其在肝癌细胞SMMC7721细胞中的表达。方法采用PCR方法获得PAPa基因;将该基因片段克隆入带有绿色荧光蛋白(greenfluorescentprotein,CFP)报告基因的真核表达载体pEGPN1中,构建融合表达载体pEGFPPAPa;通过脂质体介导的方法将该载体转染到SMMC7721中,Westernblot检测PAPa的瞬时表达,荧光显微镜检测绿色荧光蛋白表达。结果pEGFPPAPa转染SMMC7721细胞后,在细胞中检测到PAPa基因片段,Westernblot可以检测到PAPa在SMMC7721细胞表达,用荧光显微镜观察到转染细胞中有绿色荧光蛋白表达。结论pEGFPPAPa在SMMC7721细胞中获得了表达,表达的融合蛋白具有PAPa和绿色光蛋白的双重活性,该载体的成功表达是研究PAPa抗肿瘤或抗病毒活民生的基础。     

Sox9基因转染对骨髓间充质干细胞成软骨分化的影响

目的 探讨Sox9基因转染对骨髓间充质干细胞(MSCs)成软骨分化的影响.方法 采用密度梯度离心和贴壁培养法分离兔骨髓MSCs,体外培养扩增.阳离子脂质体介导重组真核表达Sox9质粒转染骨髓MSCs,通过反转录-聚合酶链反应(RT-PCR)和蛋白印迹实验(Westem blot)的方法检测基因产物的表达,荧光显微镜和流式细胞术测定细胞转染效率;四甲基噻唑蓝(MTT)法测定基因转染对细胞增殖能力的影响,流式细胞术对转基因细胞进行细胞周期分析;通过Westem blot,RT-PCR和免疫组织化学染色的方法检测Sox9基因转染对MSCs成软骨分化的影响.结果 脂质体介导Sox9基因可以成功地转染兔骨髓MSCs;Sox9基因转染对细胞增殖能力无明显影响;基因转染后的细胞表达软骨分化特异性分子Ⅱ型胶原,Sox9,aggrecan等明显增加.结论 Sox9基因转染骨髓MSCs可以获得稳定表达,基因表达产物可以促进骨髓MSCs向软骨方向的分化.     

人LIMK1基因荧光真核表达载体的构建及在人成骨肉瘤MG63细胞中的表达

目的 构建并表达人LIMK1与绿色荧光蛋白(EGFP-C1)的融合蛋白EGFP-LIMK1,并监测其在人成骨肉瘤MG63细胞内的表达及活性.方法 利用基因重组技术将LIMK1与绿色荧光蛋白融合并构建真核细胞表达质粒,通过脂质体介导法导人人成骨肉瘤MG63细胞内,用荧光显微镜观察LIMK1基因表达情况,通过Westernblot检测其在MG63细胞中的活性.结果 双酶切鉴定证实LIMK1片段已克隆到pEGFP-C1载体的Kpn Ⅰ和Not Ⅰ位点之间,测序结果证实了插入片段的正确性,转染MG63细胞后,利用荧光显微镜观察可见到绿色荧光蛋白的表达;Western blot检测证实LIMK1在MG63细胞中活性上调.结论 成功构建了含有绿色荧光蛋白基因的LIMK1真核表达载体并检测到其在真核细胞MG63中的表达及活性.     

人乙酰胆碱酯酶基因转染增强全反式维甲酸诱导胰腺癌细胞凋亡

目的探讨转染人乙酰胆碱酯酶(AChE)基因对全反式维甲酸诱导胰腺癌细胞凋亡的影响。方法应用含人AChE基因的真核表达质粒pcDNA3/AChE和绿色荧光蛋白(GFP)真核表达质粒pcDNA3/GFP,以脂质体介导上述两种质粒DNA对人胰腺癌细胞株Patu8988进行瞬时转染,转染后的细胞经全反式维甲酸(ATRA,1μmol/L)分别处理24h及48h,流式细胞仪分别检测AnnexinV染色后早期细胞凋亡率及转染效率,以细胞化学染色法(KarnovskyRoots染色)检测AChE表达。结果流式细胞仪测定GFP显示转染效率达(45.50±3.84)%,人AChE基因转染后联合ATRA治疗凋亡比例增加,转染前后分别为(14.26±1.16)%/(42.84±7.21)%(24h,P<0.01)和(18.08±0.75)%/(56.29±2.54)%(48h,P<0.01)。细胞化学染色法可见AChE阳性细胞,染色聚集于胞核部位。结论胰腺癌细胞AChE基因的高表达可增加细胞对ATRA诱导凋亡的敏感性。     

Survivin反义寡核苷酸诱导肝癌细胞凋亡的作用

目的 用反义寡核苷酸封闭肝癌细胞中survivin基因的表达,研究其诱导细胞凋亡的作用及其机制。方法 用脂质体介导survivin反义寡核苷酸转染人肝癌细胞株SMMC-7721细胞,流式细胞仪检测细胞周期变化及细胞凋亡比率,激光共聚焦显微镜观察细胞骨架微丝系统变化,激酶活性检测方法测定细胞内caspase-3活性变化,免疫沉淀法测定细胞内丝裂原活化蛋白激酶p38(p38MAPK)活性的变化。结果 脂质体介导survivin反义寡核苷酸转染肝癌细胞后Ⅰ～Ⅵ组(空白对照、正义对照、400、600、800、1000ng/ml反义转染组)细胞内p38MAPK活性分别为7.03、7.07、13.47、16.37、43.97及47.87;caspase-3活性分别为0.015±0.010、0.014±0.002、0.026±0.003、0.042±0.001、0.093±0.001及0.100±0.001;Ⅳ～Ⅵ组细胞内p38MAPK及caspase-3活性较对照组明显升高。转染后细胞发生G_2/M期阻滞,Ⅰ～Ⅵ组细胞凋亡率分别为0.70％、0.76％、2.43％、7.82％、23.11％及31.35％,各实验组细胞凋亡较对照组明显增加。细胞内微丝形态结构破坏,Ⅰ～Ⅵ组细胞肌动蛋白平均荧光强度分别为189.69±6.68、184.23±8.76、173.14±8.15、99.48±6.57、76.69±10.05及63.80±6.79,Ⅳ～Ⅵ组细胞肌动蛋白平均荧光强度较对照组明显降低。结论 脂质体介导转染survivi     

小鼠干细胞因子基因以脂质体介导转染骨髓基质细胞

目的　小鼠干细胞因子 (SCF)以脂质体介导转染骨髓基质细胞并表达。方法　采用 PCR技术从 p RC/CMV(含 SCF c DNA)中钓取SCF c DNA膜外段活性区 ,克隆入真核表达载体 pc DNA3,转染体外富集的骨髓基质细胞 ;用 RT- PCR方法检测 m RNA表达水平及通过协同刺激集落形成来检测转染细胞上清的生物学活性。结果　转染 SCF c DNA的骨髓基质细胞 SCF m RNA水平表达量高于对照组 ,其细胞培养上清协同 GM-CSF刺激骨髓细胞形成集落数比 GM- CSF单独作用高。结论　脂质体介导质粒载体转染培养富集的骨髓基质细胞并表达 ,且转染上清具有生物学活性。     

大鼠L6骨骼肌成肌细胞脂质体转染条件的研究

目的探讨脂质体介导基因转染大鼠L6骨骼肌成肌细胞的最适转染条件,同时观察绿色荧光蛋白作为报告基因用于脂质体转染条件优化的可行性.方法以绿色荧光蛋白为报告基因,通过改变质粒DNA、脂质体与质粒的比例及转染时间,在荧光显微镜下观察并计算转染效率.结果在20 mm培养皿中,1μDNA与1μl脂质体转染大鼠L6骨骼肌成肌细胞6 h,转染效率最高.结论建立了脂质体转染大鼠L6骨骼肌成肌细胞的优化转染方法;绿色荧光蛋白可作为报告基因优化脂质体转染条件.     

汉坦病毒L99株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达

从感染病毒的Vero-E6细胞中提取总RNA,采用RT-PCR和分子克隆技术将扩增到的C2糖蛋白基因插入含有CMV启动子的真核表达质粒pVAX1.通过脂质体介导转染cos-7细胞,用间接免疫荧光法检测瞬时表达的蛋白.结果获得含有编码汉坦病毒L99株包膜糖蛋白G2基因的重组质粒pVAX/C2.在转染cos-7细胞内.用IFA可检测到细胞内有特异性荧光.证实成功构建了汉坦病毒L99株包膜糖蛋白C2基因真核表达载体,并可在cos-7细胞中瞬时表达;为出血热综合征疫苗制备奠定了基础.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.