人骨髓间充质干细胞分离培养鉴定及VEGF基因的转染

目的：探讨人骨髓间充质干细胞（hMSC）分离培养、鉴定方法及人血管内皮生长因子（hVEGF）165基因转染hMSC后表达情况,拟构建血管化组织工程皮肤.方法：用密度梯度离心法分离骨髓间充质干细胞,筛选贴壁细胞培养并传代.观察细胞形态,生长情况.检验细胞CD44、CD29、CD14、CI45等表面标记.利用基因重组技术,构建pCDN3.1＋VEGF表达质粒,并将其转染第3代hMSC.经G418抗性筛选后扩大培养并用Western blot实验检测其蛋白产物.结果：骨髓密度梯度离心法分离hMSC,接种培养瓶后26 h呈对数生长,6d后达到高峰.hMSC表达CD29,CD44,不表达造血细胞标记物CD14和CD45.通过酶切鉴定验证成功构建pCDN3.1＋ VEGF表达质粒.Western blotting实验证实转染后hMSCs表达VEGF增加.结论：成功建立hMSC分离培养鉴定体系.利用基因重组技术可成功将hVEGF基因导入hMSC并产生预期效应.     

高压电击伤创面的修复

目的为了早期修复电击伤深部创面,避免并发严重感染、进行性坏死、深部组织外露和大出血,减少截肢率,最大限度地保持肢体的功能。方法早期创面彻底清创,祛除坏死病灶,有目的地保留虽变性而解剖结构尚完整的神经和肌腱,采用轴型带蒂皮瓣和肌皮瓣修复创面。结果58例皮瓣移植中仅4例皮瓣部分失活,1例皮瓣移植中断,需行截肢术,其余皮瓣成活良好,外形满意,肢体功能均有不同程度的改善。结论电击伤深部创面早期清创,应用皮瓣和肌皮瓣修复,可有效封闭创面,防止并发症,改善局部血运,保存肢体功能,降低截肢率。     

RhoA/ROCK-I信号通路与增生性瘢痕成纤维细胞结缔组织生长因子的表达

背景:前期实验表明增生性瘢痕中RhoA和ROCK-I基因表达较正常皮肤高,提示RheA/ROCK-I信号通路可能参与了增生性瘢痕的发生,但其在病理性瘢痕中的作用尚不清楚.目的:研究RhoA/ROCK-I信号通路在增生性瘢痕成纤维细胞结缔组织生长因子(connective tissue growth factor,CTGF)表达调控中的作用.方法:分离培养人增生性瘢痕组织来源的成纤维细胞,应用转化生长因子β1及Rho激酶的特异抑制剂Y-27632对细胞进行干预实验.采用实时荧光定量PCR及免疫荧光细胞化学方法检测瘢痕成纤维细胞中RhoA,ROCK-I及CTGF mRNA与蛋白的表达.结果与结论:给予转化生长因子β1后,增生性瘢痕成纤维细胞中RheA,ROCK-I及CTGF mRNA与蛋白表达明显增多(P<0.01):而Y-27632能阻碍转化生长因子β1的作用;但单独给予Y-27632并不引起瘢痕成纤维细胞中RhoA,ROCK-I及CTGF的mRNA与蛋白表达改变.说明转化生长因子β1可通过RhoA/ROCK-I信号通路调控CTGF mRNA与蛋白的表达,即RhoA/ROCK-I信号通路参与了瘢痕成纤维细胞CTGF的表达调控,阻断RheA下游通路是增生性瘢痕治疗靶点之一.     

正天丸治疗头痛临床研究

正天丸是首创于1985年的中成药复合组方,由钩藤、川芎、白芍、当归、地黄、白芷等15味中草药组成,具有疏风活血、养血平肝、通络止痛的功效,广泛适用于多种头痛.笔者将正天丸上市至今20余年有关临床研究总结分析如下.     

甘露醇对新西兰兔严重烧伤早期血清TNF-α和IL-6的影响及其脏器功能的保护机制

目的探讨甘露醇补液疗法对严重烧伤新西兰兔组织器官的保护作用及其保护机制。方法将12 只新西兰兔构建30%
TBSA深度烧伤模型后,随机分为对照组和甘露醇组,烧伤后1 h开始补液治疗,分别于烧伤前、烧伤后1、4、8、24、48 h收集血清
及48 h 总尿液,用夹心酶联免疫吸附法测定血清、尿中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）含量;生化法测定ALT、
AST、GGT、CK、CK-MB、BUN和Cr 含量。结果烧伤后1 h 两组兔血清TNF-α和IL-6 开始升高,伴随ALT、AST、GGT、CK、
CK-MB、BUN和Cr升高;与对照组相比,甘露醇组兔48 h尿液TNF-α和IL-6总排泄量较高（P<0.05）,血清TNF-α和IL-6水平则
较低（P<0.05）,ALT、AST、GGT、CK、CK-MB、BUN和Cr含量也较低（P<0.05）。结论甘露醇补液疗法可降低烧伤早期血中炎
症介质TNF-α和IL-6的含量,减轻心、肝、肾的功能损害。
     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

益气活血汤对脑分水岭梗死急性期多时点NIHSS,中医证候,HSP-70的影响

目的:探讨益气活血汤对脑分水岭梗死急性期患者多时点美国国立卫生研究院卒中量表(NIHSS)、中医证候、热休克蛋白-70(HSP-70)的影响,及HSP-70的动态变化与疾病发展和中医证候演变的相关性。方法:脑分水岭梗死发病72 h以内的137例患者随机分为治疗组69例,对照组68例。治疗组在对照组治疗的基础上,口服中药益气活血汤。发病3 d内、第7,14天记录NIHSS评分,进行中医证候评价。采集HSP-70,进行比较及相关性分析。结果:治疗后治疗组NIHSS差值高于对照组(P0.05),治疗组气虚和血瘀的改善显著优于对照组(P0.01)。两组发病3~7 d,治疗组HSP-70升高幅度高于对照组(P0.05)。两组发病7~14 d,治疗组HSP-70下降幅度高于对照组(P0.05)。3个时点HSP-70与NIHSS,气虚,血瘀均成负相关(P0.05)。发病3 d内、第7天NIHSS与气虚、血瘀均成显著正相关(P0.01)。发病第14天NIHSS与气虚成正相关(P0.05),与血瘀成显著正相关(P0.01)。结论:益气活血汤有助于改善脑分水岭梗死急性期患者的神经功能缺损情况,对气虚血瘀的中医证候有显著改善作用。益气活血汤可能有利于HSP-70发挥细胞保护功能。因其高度相关性,发病后HSP-70的表达水平在不同时间点的差异与关联能够从微观角度反映脑分水岭梗死急性期患者的神经功能缺损的变化规律及中医证候的演变趋势。     

从络病论治紧张性头痛

络脉是气血会聚之处,具有贯通营卫、环流经气、渗透气血、互化津血的生理功能,是内外沟通的桥梁,有气络与血络之分。紧张性头痛病位在气络与血络,初病中络,以邪气阻络、气机不畅为主,多为络之气病;病久不愈,络脉瘀阻,渐成痼结,则气病及血。主要病因病机为神形过用而致的心肝失调,气血失和,络脉瘀滞。络病易滞易瘀,气络之滞起于肝,血络之瘀因于心。紧张性头痛反复发作,缠绵难愈,其疼痛特点是痛处固定,常伴有颈部、颞肌压痛,切合"久痛入络"之说。调心疏肝、疏畅气血、辛散通络为基本治法。气络之病要在疏肝,血络之病重在调心。临证应重视风药的运用,其味多辛,其性主散,辛散轻扬,性主上浮,疏通气血,兼具引经、止痛之效,为治疗紧张性头痛之良药。     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.     

miR-196a-5p抑制小鼠胚胎干细胞的自我更新

目的 研究miR-196a-5p在小鼠胚胎干细胞(mESC)自我更新中的功能.方法 定量PCR法确定miR-196a-5p在mESC早期分化过程中的表达.在胚胎干细胞中过表达miR-196a-5p类似物,通过集落形成实验、细胞周期分析、碱性磷酸酶染色和Oct4染色确定miR-196a-5p过表达对mESC自我更新及增殖的影响.结果 miR-196a-5p在mESC早期分化过程中表达上升(P＜0.05),在mESC中过表达miR-196a-5p类似物后,ESC的集落形成及增殖能力被明显抑制(P＜0.01),碱性磷酸酶活性下降,Oct4蛋白水平也出现明显下降.结论 miR-196a-5p具有抑制mESC自我更新的能力,在mESC早期分化过程中可能发挥重要作用.