Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
In this article, three research cases are introduced to show the rationale of analysis of papers cited in evidence-based guidelines and the implementation steps. The ultimate goal of medical research is to promote health; evidence-based guidelines are of most importance to medical practice, which can be directly used in clinical practice and health prevention. One of the ways in which research can influence practice is through its contribution to being references of guidelines, and it’s a new way to evaluate biomedical research’s “payback” on health.
Objective To investigate the effects of intermittent negative pressure on the mRNA expression of osteoprotegerin (OPG) and osteoprotegerin l igand (OPGL) in human BMSCs cultured in vitro. Methods BMSCs were isolated from adult marrow donated by 2 hip osteoarthritis patients with prosthetic replacement in January 2008 and cultured in vitro. The third passage cells were divided into experimental group and control group. The experimental group was induced by negative pressure intermittently for 2 weeks (pressure: 50 kPa, 30 minutes each time, twice per day) and the control groupwas routinely cultured. After 2 weeks of culture, cell morphology was observed by inverted phase contrast microscope, and the mRNA expressions of OPG and OPGL in BMSCs were analyzed by real-time PCR. Results The cell prol iferation speed of the experimental group was slower than that of the control group. The cell morph changed from shuttle to megagon with some prominences in experimental group and the cell morph kept shuttle in the control. The mRNA expression of OPG in experimental group increased significantly (P lt; 0.01) and the mRNA expression of OPGL in experimental group decreased significantly compared with control group (P lt; 0.01) 2 weeks later. Conclusion Intermittent negative pressure is capable of promoting the expression of OPG, while inhibiting the expression of OPGL in human BMSCs.
To detect the cell density, apoptotic incidence and the expressions of Bax and Caspase-3in human lumbar intervertebral discs, so as to further understand the mechanism of human lumbar intervertebral discdegeneration and provide a new idea for biologic treatment of it in future. Methods From May to December in 2006,30 human lumbar intervertebral discs in experimental group(L2 to S1)were surgically collected from 27 patients undergoing posterior lumbar intervertebral discoidectomy and fusion. All the cases were affirmed by MRI and they never experienced discography, collagenolysis of nucleus pulposus and percutaneous laser disc decompression. The control group consisted of 20 human lumbar intervertebral discs(L2 to S1)harvested from 5 young men without spine-related condition immediately after their accidental death. Apoptotic disc cells were detected by TUNEL and histomorphology, and immunohistochemical staining with SP method was performed to examine the expressions of Bax and Caspase-3 in all specimens. Results HE staining disclosed that the average cell density in control group (17.16 ± 1.22)/HP was higher than that in experimental group (12.41 ± 0.95)/HP (P lt; 0.01). However, TUNEL staining observed that the average TUNEL positive incidence in control group (6.97% ± 0.92%) was lower than that in experimental group (12.59% ± 0.95%), (P lt; 0.01). Immunohistochemical staining with SP method showed that the Bax and Caspase-3 positive incidence of nucleus pulposus in control group (11.02% ± 1.18%, 9.01% ± 1.00%) were lower than those in experimental group (19.29% ± 1.18%, 15.07% ± 0.97%), (P lt; 0.01). The results of the average gray scale value of nucleus pulposus in control group were 187.33 ± 7.88 and 185.68 ± 3.26, respectively, with 124.98 ±6.69 and 160.13 ± 4.37 in experimental group. There was significant difference between the two groups (P lt; 0.01). When thetotal 50 specimens in the two groups were analyzed, TUNEL positive incidence showed significant inverse correlations with their respectively corresponding cell densities (r = － 0.88, r = － 0.93, P lt; 0.01). The Bax and Caspase-3 positive incidence of nucleus pulposus showed significant positive correlation with the TUNEL positive incidence of nucleus pulposus (r = 0.83, r = 0.91, P lt; 0.01). Conclusion The decrease of cell density is involved in the development of human lumbar intervertebral disc degeneration. Bax and Caspase-3 might play a role in disc cell apoptosis in nucleus pulposus of human lumbar intervertebral disc.
目的进一步提高Ebstein心脏畸形外科治疗的效果，减少并发症，降低死亡率。 方法回顾性分析河南省胸科医院2010年1月至2013年3月10例Carpentier B型Ebstein心脏畸形患者行改良Carpentier术的临床资料，其中男5例、女5例，年龄15～47（25.0±6.2）岁；血氧饱和度 ＜90%者4例，＞91%者6例。手术在全身麻醉、低温体外循环下进行，术中首先探查清楚右室内结构及三尖瓣叶发育情况，确认有发育良好的三尖瓣前瓣，而后由内向外双层连续缝合纵形折叠房化右心室。旷置三尖瓣隔瓣及后瓣；根据患者年龄、体重选择适当型号的人工瓣膜成形环，间断缝合固定成形环；注水检查有无反流，根据情况再做适当修理，至注水无反流为准。其中2例患者右心室发育差，术毕中心静脉压达15 cm H2O，加行双向格林（Glenn）手术。 结果术中应用31号成形环1例，29号成形环6例，27号3例。主动脉阻断时间45～66（51.5±2.6）min。术后循环均平稳，无一例死亡，无低心排血量综合征发生。术后心脏彩色超声检查：三尖瓣无反流6例，轻度反流4例；心功能Ⅰ级6例，Ⅱ级4例。术后随访2个月至2年，1例患者1年后出现三尖瓣中量反流，强心利尿治疗后好转；余9例心功能（NYHA分级）Ⅰ级8例，Ⅱ级1例。 结论改良Carpentier术治疗B型Ebstein心脏畸形效果良好，方法简单实用，易于推广。
ObjectiveTo systematically review the efficacy of early physical rehabilitation for patients with stroke-induced hemiplegia. MethodsDatabases including PubMed, Web of Knowledge, The Cochrane Library (Issue 7, 2014), EMbase, CNKI, VIP and WanFang Data were searched to collect randomized controlled trials (RCTs) about early physical rehabilitation for patients with stroke-induced hemiplegia from inception to 1st August 2014. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Then, metaanalysis was performed using RevMan 5.2 software. ResultsA total of 6 RCTs were included. The results of metaanalysis showed that, after 1 month of treatment, the Fugl-Meyer scores (MD=18.02, 95%CI 6.24 to 29.80, P=0.003) and BI scores (MD=32.32, 95%CI 24.28 to 40.36, P<0.000 01) in the early physical rehabilitation group were higher than that in the conventional rehabilitation group. But after 3 months of treatment, there were no statistical differences between two groups in the proportion of patients with 0-2 MRS scores, mortality and the incidence of complications. ConclusionCurrent evidence shows that early physical rehabilitation can effectively improve the limb motor function and daily living ability in patients with stroke-induced hemiplegia. However, due to the limited quantity and quality of the include studies, more large-scale, high quality RCTs are needed to verify the above conclusion.
目的 总结法洛四联症矫治术的临床经验，进一步提高治愈率，降低其并发症和死亡率。 方法 回顾性分析2005年4月1日至2013年3月31日河南省胸科医院704例行法洛四联症矫治术患者的临床资料，其中男394例、女310例，年龄3个月至45岁，平均（3.6±6.6）岁。 结果 684 （97.20%） 例治愈，死亡20例，死亡率2.8%。死于低心排血量综合征和多器官功能衰竭16例，急性肾功能衰竭2例，术后灌注肺合并肺部感染1例，心搏骤停1例。因术后出血量多，再次开胸止血21例（2.98%）；脱离呼吸机后二次气管内插管21例（2.98%），腹膜透析治疗肾功能不全25例，发生感染性心内膜炎10例，Ⅲ°房室传导阻滞2例；10例术后出院前复查发现小的室间隔缺损残余漏，5例跨肺动脉瓣压差超过40 mm Hg。随访1～8年，随访到658例，随访率98%。随访期间因心衰死亡3例，因心内膜炎死亡1例。 结论 术前精确诊断，把握好手术适应证，术中矫治满意，术后及时恰当处理是手术成功的关键。