HSV-TK自杀基因对涎腺多形性腺瘤细胞治疗的实验研究

目的研究单纯疱疹病毒胸苷激酶基因(Herpes simplex virus thymidine kinase，HSV-TK)／丙氧鸟苷(ganciclovir，GCV)系统对人涎腺多形性腺瘤体外基因治疗的作用。方法采用腺病毒包装重组脂质体介导的含有HSV-TK全长的cDNA真核表达质粒PDC312-HSVTK转染人涎腺多形性腺瘤细胞，通过RT-PCR检测TK基因在转染细胞中的表达；用MTT法检测HSV-TK／GCV系统对多形性腺瘤细胞的杀伤作用和旁观者效应：采用倒置显微镜、组织学染色等观察HSV-TK／GCV系统作用细胞后的形态学改变。结果HSV-TK基因转染24小时后，肿瘤细胞开始出现空泡性变；转染48小时后，RT-PCR从转染的细胞中成功扩增出1150bp的特异性TK基因片段。转染36小时后加入不同浓度的GCV治疗，细胞出现核固缩，胞浆裂解，随之细胞死亡、脱壁的现象。细胞存活率随HSV-TK／GCV作用时间延长而逐渐下降，当加入10^-4mol／LGCV治疗7天时，HSV-TK／GCV系统的杀伤作用最强，细胞存活率为10．3％。当TK阳性的细胞占细胞总数50％时，其旁观者效应最明显，细胞存活率为31．7％。结论HSV-TK／GCV系统对涎腺多形性腺瘤细胞具有明显的杀伤作用和旁观者效应。     

氯化镧对人牙龈成纤维细胞增殖的影响

目的:检测不同浓度氯化镧(La3 )对人牙龈成纤维细胞(HGF)增殖的影响。方法:体外培养HGF,用四唑盐法(MTT)检测6种浓度La3 (1×10-6、1×10-5、5×10-5、1×10-4、1×10-3及1×10-2 mol/L)对HGF的作用。结果: 1×10-2 mol/L的La3 能抑制HGF的生长, 1×10-6、1×10-5、5×10-5、1×10-4及1×10-3 mol/L的La3 对HGF生长无抑制作用。结论: 1×10-2 mol/L浓度的La3 对HGF增殖有抑制作用, 1×10-4 mol/L以下浓度的La3 对HGF无抑制作用。     

促结缔组织增生型成釉细胞瘤细胞的体外培养与鉴定

目的：观察体外培养的促结缔组织增生型成釉细胞瘤细胞的生长特点。方法：对促结缔组织增生型成釉细胞瘤细胞用酶消化法进行体外培养，免疫组化证实其细胞来源，以倒置显微镜观察细胞形态及生长情况．并用流式细胞仪分析细胞周期，MTT法绘制细胞生长曲线。结果：促结缔组织增生型成釉细胞瘤为上皮来源，细胞在体外培养过程中包含2种形态的细胞，与实性成釉细胞瘤细胞在体外培养相比，存在较多梭形细胞，细胞生长缓慢，培养的各代细胞未见异倍体。结论：促结缔组织增生型成釉细胞瘤细胞在体外培养过程中包含较多的梭形细胞．可能与其较多的细胞间质有关。     

Establishment of cell free conversion system with biotin-labelled recombinant PrPsen expressed in E. coli

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.     

Cell kinetics of mechanically stimulated rat oral epithelia

OBJECTIVE: To study cell kinetics of rat gingival (GE), sulcular (SE) and junctional (JE) epithelia in the steady-state and after application of mechanical pressure. DESIGN: Elastic bands were inserted between first and second maxillary molars of 8-week-old male rats, which were labelled with H(3) TdR and killed in groups of six to seven animals together with equal-sized groups of labelled control animals at intervals between 1 and 168 h. Autoradiographs were used to determine epithelial cell proliferation on the pressure side of M1 by calculating the percentage of (3)H TdR-labelled cells (PLC) in the basal (BL) and suprabasal (SL) layers of GE, SE and JE and to estimate median cell cycle (MCC) duration of BL cells by plotting mean and median grain counts against time. RESULTS: (3)H TdR-labelled cells were present in SL of SE and JE 1-12h after isotope injection suggesting that the BL might be not the only source of progenitor cells for JE as they might also be derived through migration from adjacent SE. Application of pressure significantly (ANOVA, P<0.05) reduced PLC in BL of GE, SE and JE indicating a decrease of cell proliferation after 1-12h in response to pressure. In steady-state, the MCC durations of BL cells of GE, SE and JE were 39, 14 and 9h, respectively. After application of pressure, they increased significantly (chi(2)-test, P<0.05) to 48, 44 and 34 h, respectively. CONCLUSIONS: Sustained pressure may lead to reduction of proliferative activity of these epithelia inducing slower progression of progenitor cells through the cell cycle.     

5DF血球分析仪检测白细胞分类计数与瑞氏染色镜检结果对比研究

目的　探讨5DF血球分析仪白细胞分类计数与传统的瑞氏染色镜检计数结果之间的差异。方法　用5分类血球分析仪和瑞氏染色法同时对10 0份血样进行分类计数,并用统计学分析研究。结果　中性粒细胞、嗜酸、嗜碱性细胞和淋巴细胞分类计数两法无明显差异(P均>0 .0 5 ) ,但单核细胞分类计数仪器法比镜检法结果偏高(P <0 .0 0 1)。结论　对于一般血液常规检查可选用仪器法筛查,而血液系统疾病或某种原因引起的细胞结构改变应选用镜检法。     

Effects of prenatal chronic mild stress exposure on hippocampal cell proliferation,expression of GSK-3α, β and NR2B in adult offspring during fear extinction in rats

Stress during pregnancy has been implicated as a risk factor for the development of many mental disorders; however, the influence of prenatal stress on the fear or anxiety-related behaviors, especially the fear extinction in adult offspring has been little investigated. In order to investigate how prenatal stress affects fear extinction, which is regarded as a form of new learning that counteracts the expression of Pavlovian's conditioned fear, a rat model of prenatal chronic mild stress (PNS) was used to evaluate the effects of PNS on fear extinction in adult offspring. The expression of hippocampal glycogen synthase kinase-3s (GSK-3α, β), N-methyl-d-aspartic acid receptors (NMDARs)-2B and the hippocampal cell proliferation in dentate gyrus in the adult offspring during fear extinction were studied. Our results showed that PNS significantly reduced body weight of pups, indicating PNS might induce growth retardation in offspring. Moreover, PNS significantly enhanced the freezing behavior of offspring at the phase of extinction, suggesting PNS impaired the abilities of fear extinction learning. In addition, PNS significantly increased the levels of GSK-3α, β and NR2B, but reduced hippocampal cell proliferation during fear extinction. Taken together, our findings suggest that maternal stress during pregnancy can impair the fear extinction of adult offspring, probably by affecting the neural plasticity of brain.     

Cell transplantation therapy for a rat model of secondary lymphedema

Background

Although lymphedema is a progressive and lifelong condition, substantial advances in therapeutic intervention are limited. The development of a novel therapy for lymphedema is urgent for those patients suffering from it. The aim of this study was to investigate the usefulness of a new cell transplantation therapy in the rat tail model of secondary lymphedema.

Materials and methods

We prepared two cell sources, human dermal microvascular endothelial cells (HDMECs) and lymphatic endothelial cells (LECs), which were collected from the resected normal dermis of patients with breast cancer. After the animal model of secondary lymphedema of the nude rats' tails was established, phosphate-buffered saline, purified LECs, or unpurified HDMECs were injected in the rats' tails five times for more than 14 d. The evaluations were performed by measuring the circumference, fluorescence lymphography, and histologic analysis of the rats' tails between each group.

Results

The isolated cells by the simple immunomagnetic sorting from HDMECs were positive for a pan-endothelial marker (CD31) and lymphatic-specific markers (podoplanin, lymphatic vessel endothelial hyaluronan receptor-1 [LYVE-1], and prospero homebox 1 [Prox-1]), and were considered to be LECs. In the cell transplantation group, which was injected with human LECs, the circumference, lymphatic flow, and thickness of the skin of the rat tail became thinner than the groups injected with unpurified HDMECs or phosphate-buffered saline. Immunohistochemistry of the rat tails showed that the number of own lymphatic vessels was increased in the purified LEC transplantation group compared with the other groups. Furthermore, in the LEC transplantation group, some vessels were immunopositive for human-podoplanin or -LYVE-1 and the areas adjacent to the vessels were rat-podoplanin or -LYVE-1 immunopositive.

Conclusions

Our findings indicate that cell transplantation therapy using human LECs improved the secondary lymphedema in the nude rat tail. This therapeutic strategy may merit clinical investigation in patients with lymphedema.