应用3H-TdR掺入法对SMMC-7721细胞株药敏试验条件的探讨与应用

目的探讨3H-TdR掺入法在进行SMMC-7721系细胞肿瘤药敏试验实验时的最佳实验条件.方法确定出3H-TdR掺入法药敏实验时的最适细胞浓度、最适实验药物浓度、3H-TdR掺入最适时间;在最适条件下采用3H-TdR掺入法测定肝癌SMMC-7721细胞株对临床常用的9种抗癌药物的敏感性.结果应用3H-TdR掺入法检测肝癌SMMC-7721细胞株药敏试验的最适实验细胞浓度为1×104个/孔,最适试验药物浓度为1×PPC,3H-TdR最适掺入时间为收集细胞前8 h;肝癌SMMC-7721细胞株对DDP、ADM、5-FU、CPT高度敏感,对MTX、VP-16、MMC、NVB低度敏感,对PYM不敏感.结论 3H-TdR掺入法可用于肿瘤药物敏感性测定并确定出它的最适实验条件.     

大鼠脂肪干细胞复合胶原-壳聚糖-硫酸软骨素三维支架构建组织工程软骨

Objective To evaluate the character of the collagen-chitosan-chondroitin sulfate scaffold seeded with rat adipose tissue-derived stromal cells. Methods A dipose tissue were harvested from 6 weeks old Wistar rats and the stromal cells were harvested by type Ⅰ collagenase and then cultured in vitro. Type Ⅰ collagen was fully mixed with chitosan, freeze-dried and cross-linked with chondroitin sulfate, then freeze-dried again and sterilized by ethylene oxide. The pore diameter, water content, porosity of the scaffold were tested. The adipose tissue-derived stromal cells were digested, seeded into the plates, scaffold, and cen-trifuged into pellet, and then induced into cartilage. MTT detection for cell proliferation was done. After 3 weeks, the cell morphology, and cell proliferation and adhesion were observed, and chondrngenic differenti-ation was also analyzed. Results The pore diameter, water content, porosity tested for the scaffold showed an appropriate form. Cell proliferation showed faster in the scaffold and pellet culture system after 5 day, there was still cell proliferation in the scaffold system after 14 days but no obvious changes in the pellet cul-ture system; ceils on the scaffold proliferated densely showed by histological staining, but there was a scaf-fold structure residues in the inner layer. The finding of type Ⅱ immunohistochemistry stain showed that cells express strong positive for type Ⅱ collagen in the scaffold and pellet culture system whereas it was weakly positive in the plate culture system; the specific mRNA for cartilage, type Ⅱ collagen, aggrecan and SOX-9 were expressed in all three systems showed by RT-PCR, but type X collagen was expressed continu-ously in the plate culture system and expressed after 21 days in the pellet culture system, whereas it was not detected in the collagen-chitosan-chondroitin sulfate scaffold system. Conclusion The parameters of the collagen-chitosan-chondroitin sulfate scaffold were suitable in our study. The results suggested that it can promote the adipose tissue-derived stromal cells proliferation and chondrogenic differentiation better than the plate and pellet culture systems and maintain the phenotype of chondrocytes well; it is the optimal choice for cartilage tissue engineering in the future.     

福田4500型B超软故障检修与分析

通过福田4500型B超软故障检修与分析，介绍了该机故障检修分析过程及造成该故障的原因。     

罗格列酮对多囊肾囊肿衬里上皮细胞p38促分裂原活化蛋白激酶信号通路的影响

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells (PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone (10 μmol/L) +GW9662 (10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone(10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phuspho-p38 (p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos anti c-jun compared with control group (P<0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P<0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P<0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independcnt.     

人resistin基因原核表达载体构建和表达

[目的]构建人resistin基因原核表达载体并观察其表达,为开展对resistin的研究打下实验基础.[方法]酚氯仿一步法从一位2型糖尿病合并胆管癌患者腹壁皮下脂肪组织抽取总RNA,RT-PCR法扩增出re-sistin基因cDNA,经测序后,PCR产物亚克隆入T载体,用EcoRI和KpnI分别酶切重组T质粒和原核表达载体pGENKS,然后resistin基因片段和表达载体通过T4DNA连接酶进行定向连接,用核酸内切酶EcoRI、KpnI酶切和测序方法鉴定出重组表达质粒.重组表达质粒转化大肠杆菌JM109,经1 mmol/LIPTG在32℃诱导表达2 h,裂解细菌,进行PAGE凝胶电泳鉴定融合蛋白的表达.[结果]RT-PCR扩增产物分子大小为327 bp,序列分析表明为人resistin基因完整编码区.PCR产物与T载体连接、转化大肠杆菌后,从细菌质粒中酶切回收了目的片段.重组表达质粒经EcoRI和KpnI双酶切后能产生出327 bp、4 980 bp大小的两个片段,序列分析表明重组表达质粒包含人resistin基因完整编码区,基因编码无移位,PAGE凝胶电泳表明在细菌裂解上清有分子质量为39.5 kD的融合蛋白.[结论]成功构建了人resistin基因原核表达载体,且构建的载体能表达出含目的蛋白的融合蛋白,为下一步研究打下了实验基础.     

阴道毛滴虫Rab11鸟苷三磷酸酶eDNA克隆和序列分析

目的 近年研究表明Rab11鸟苷三磷酸酶在调节各种真核细胞的膜转运通道中发挥着重要作用。但是，对于原生动物毛滴虫膜转运系统的研究仍甚少。本研究旨在克隆和分析阴道毛滴虫Rabll鸟苷三磷酸酶基因，以便进一步探讨其功能。方法 使用SMART cDNA文库构建试剂盒构建阴道毛滴虫cDNA表达文库，用生物信息学进行TvRab11同源分析，鉴定TvRab11基因，用PCR方法扩增基因组DNA，TA克隆TvRab11 cDNA、测序及序列分析。结果 在分离出的cDNA克隆中发现一个Rab11鸟苷三磷酸酶同源基因。该cDNA序列长710bp，读码框含636bp，推测蛋白质序列具211个氨基酸。序列分析表明该基因系Rab11亚家族的同源基因，其推测肽链与拟南芥Rab11c的同源性晟高（一致性60％，相似性79％），与人类Rab11b次之（一致性58％，相似性78％）。该氨基酸序列拥有Rab鸟苷三磷酸酶家族的所有保守结构域和特异性Rab结构域（RabF1-5）。进化树分析也表明该基因系Rab11的同源基因。序列分析还显示该基因的基因组DNA序列与cDNA序列完全一致．提示该基因无内含子。结论分析表明该基因是阴道毛滴虫Rab11鸟苷三磷酸酶同源基因，其功能有待进一步研究。     

前路一期病变椎体切除并重建治疗胸腰椎结核并后凸畸形

目的:观察前路一期病变椎体切除、人工椎体或钛网融合器植骨替代、椎体钉板或钉棒系统内固定治疗连续两个及以上节段胸腰椎结核并后凸畸形的疗效。方法:34例病变累及连续两个及两个以上椎节的胸腰椎结核患者,术前后凸Cobb角27.8°￣65.4°(38.6°±10.3°),一期行前路病变椎体切除,椎间撬拔撑开复位,人工椎体或钛网融合器植骨替代,辅以椎体钉板或钉棒系统短节段邻近椎节内固定,重建脊柱稳定性,术后均给予短疗程化疗。观察术后局部疼痛缓解、脊髓神经功能恢复、后凸畸形矫正及脊柱稳定性情况。结果:患者术后局部疼痛缓解,术前伴有脊髓神经损伤的12例患者术后神经功能均有不同程度恢复。影像学检查示脊柱内固定物位置良好,椎体序列恢复良好,椎间高度恢复。后凸Cobb角矫正至2.1°￣14.2°(7.5°±8.3°),平均矫正31.2°±8.5°。随访18￣54个月,平均35个月。末次随访时后凸矫正度丢失4.3°±3.8°,均无结核复发。结论:连续两个及两个以上节段的胸腰椎结核采用前路一期行病变椎体切除有利于病灶彻底清除,减少复发;也有利于椎管彻底减压。前路椎体替代、植骨内固定重建脊柱稳定性可更好地纠正和预防脊柱后凸畸形。     

基于形状因子分析的重叠细胞自动判别技术研究

重叠细胞的判别在细胞计数和参数测量中有非常重要的意义.本文根据重叠细胞的形态特征,提出根据形状因子来判断细胞是否重叠.首先研究了形状因子的计算方法,对重叠细胞形状因子的阈值P0进行了探讨和实验,得出P0=0.5:当PE≤P0,认为目标存在细胞重叠;PE＞P0,认为目标不存在细胞重叠.实验结果证明本技术能检测出不同形状的重叠细胞,准确率为95%.     

微嵌合体表达与移植肾免疫耐受的实验研究

目的探讨肾移植术后受者微嵌合体表达与免疫耐受的相关性。方法采集接受男性供体肾脏移植术后女性受者不同生存期外周血及尿沉渣标本,采用Y染色体三对引物SRY1、DYZ11st和DYZ12nd,应用PCR和RT-PCR方法检测标本中微嵌合体特异性代表Y染色体DNA和mRNA的表达,同时测定移植肾功能。结果在130例供者为男性的女性肾移植受者检测到嵌合阳性者98例,占75.39%(n=130),嵌合阴性者32例,占24.61%。嵌合阳性与阴性两组比较,移植肾平均存活时间分别为(8.7±3.5)年和(5.4±3.3)年,P<0.05;嵌合阳性者发生过排斥反应的有11例,占11.22%,而嵌合阴性者为9例,占28.13%,P<0.05;血清肌酐在嵌合阳性者(74.3±32.5)mol/L,而嵌合阴性者为(113.6±37.8)mol/L,P<0.05。结论受者体内嵌合阳性与阴性者相比具有更好的移植肾功能和低排斥率,肾移植术后受者生存期愈长嵌合阳性率愈高,提示肾移植术后受者体内的嵌合状态与免疫耐受具有相关性。     

血管内覆膜支架置入治疗降主动脉夹层动脉瘤

目的 :介绍血管内覆膜支架置入治疗降主动脉夹层动脉瘤的初步经验。材料与方法 :本组 6例 Debakey 型的慢性降主动脉夹层动脉瘤患者行血管内覆膜支架置入治疗。经股动脉将 Talent覆膜支架置于夹层动脉瘤裂口处 ,支架张开使覆膜支架固定于裂口附近的主动脉壁上将裂口封闭并阻断血流。结果 :6例病人均获得成功 ,手术和临床成功率 10 0 %。 1例患者术中出现内漏 ,置入第二枚支架后漏口封闭。平均随访 7个月 ,所有患者内膜裂口全部完全封闭 ,假腔内血栓形成无内漏 ,假腔均明显缩小。结论 :血管内覆膜支架置入治疗主动脉夹层动脉瘤具有安全可靠 ,患者术后恢复快的优点