不同分离方法与培养条件下大鼠骨髓间充质干细胞生长增殖情况比较

目的:应用贴壁分离法和密度梯度离心法以不同培养条件观察纯化过程中对获得的骨髓间充质干细胞的影响,试图寻找获得高质量、高活性的骨髓间充质干细胞的培养条件。方法:实验于2005-09/2006-01在解放军第四军医大学实验动物中心实验部进行。①SPF级雄性Wistar大鼠18只,贴壁分离培养取12只,根据血清和培养基的不同分为4组:进口血清 DMEM组、进口血清 F12-DMEM组、国产血清 DMEM组、国产血清 F12-DMEM组,3只/组。密度梯度离心培养实验取剩余6只,以分离液的不同分为Ficoll分离液组、Percoll分离液组,3只/组。②骨髓取材:各组大鼠断颈处死后,无菌取双下肢,剔除股骨、胫骨周围肌肉组织,剪去骨干的两端,暴露骨髓腔,穿刺两侧骨端取出骨髓。③贴壁分离法:进口血清 DMEM组、进口血清 F12-DMEM组、国产血清 DMEM组、国产血清 F12-DMEM组大鼠分别采用相应的血清和培养基冲洗骨髓,制备单细胞悬液,接种于培养瓶中,5d后首次更换培养液,弃去未贴壁细胞,此后每3～4d换液1次。④密度梯度离心法:Ficoll分离液组选用的Ficoll分离液密度为1.077;Percoll分离液组将Percoll分离液与0.1mol/L磷酸盐缓冲液按9∶1比例混匀,密度为1.073。将两组大鼠骨髓置入离心管,离心弃上清,轻轻叠加到相同体积的各自对应分离液上,再次离心收集界面层白色混浊液,采用F12-DMEM培养液重悬细胞,按1×109L-1的密度接种于培养瓶中,培养条件与贴壁分离法相同。⑤指标检测:倒置显微镜下,每天观察贴壁分离、密度梯度离心不同培养条件下细胞的生长情况和活体形态特征。进行锥虫蓝排斥试验,蓝染细胞为死亡细胞,在3min内用计数板分别计数活细胞和死细胞,未被蓝染细胞所占细胞总数的百分比即为初步得到细胞活性的数据。两种分离方法均取生长良好的第3代细胞,以时间为横坐标,细胞数为纵坐标,绘制生长曲线。结果:18只大鼠均进入结果分析。①细胞形态观察结果:贴壁分离法:采用进口血清培养的两组细胞形态学上都表现为长梭形,细胞活性较高,且给予F12-DMEM培养基的细胞增殖较快,集落融合较早,传代所需时间短;采用国产血清培养的两组细胞中,给予F12-DMEM培养基可以获得梭形细胞,而给予DMEM培养基后则多为类圆形骨髓样细胞,仅见极少梭形细胞。密度梯度离心法:Ficoll分离液组和Percoll分离液组均可培养出梭形骨髓间充质干细胞,细胞活性均高,但集落融合、传代所需的时间均较贴壁分离法长。②细胞活性检测结果:贴壁分离法:进口血清 DMEM组、进口血清 F12-DMEM组、国产血清 DMEM组、国产血清 F12-DMEM组的活细胞率分别为98.3%,98.7%,97.1%,97.7%,组间比较差异无显著性意义(χ2=0.054～0.620,P均>0.05)。密度梯度离心法:Ficoll分离液组活细胞率与Percoll分离液组相似(96.9%,97.1%,t=1.066,P>0.05)。③第3代细胞生长曲线测定结果:不同培养条件下各组第3代细胞生长曲线基本相似,在培养1d时,细胞量稍有减少;4d后细胞数均迅速增长;8d进入平台期,细胞增殖减慢。结论:采用全骨髓贴壁分离法和密度梯度离心法,只要选择合适的培养条件均可获得骨髓间充质干细胞,但选用进口胎牛血清、F12-DMEM培养基效果较好。     

Asbestos — A hazard to the community

During a recent study of pulmonary fibrosis in one clinic area in Glasgow 21 proven cases of lung pathology associated with exposure to asbestos have come to light.

Difficulties in diagnosis may be due to “patient error” in that the patient may not remember that he had been in contact with asbestos, or know that he is exposed to asbestos in his current occupation — a joiner, for example, sawing up asbestos boards.

Examples are given of asbestosis or pleural tumours occurring many years after a short period of exposure to the dust.

Persons in innocent-looking occupations, such as joinery, may be in great danger from working near persons like laggers, who use asbestos. Especially in confined spaces on shipboard.

The importance is stressed of taking a very full occupational history, from the time the patient left school, where there is obscure pulmonary fibrosis.

The value of open lung biopsy is shown in diagnosing doubtful cases of early asbestosis.

The impression is gained that the incidence of asbestosis is increasing, and that it may come to assume great public health importance owing to the increased importation and more widespread use of asbestos.

The proposal to mix asbestos with tarmacadam for road use is viewed with concern.     

Over-expressions of AMPK subunits in ovarian carcinomas with significant clinical implications

ABSTRACT: BACKGROUND: AMP-activated protein kinase (AMPK) has recently been considered as a potential target for cancer therapy. However, the expression status of various subunits of the heterotrimeric AMPK in human cancers is rarely reported. We decided to determine their expressions in ovarian carcinomas and their relationships with the disease. METHODS: Expressions and locations of the AMPK-alpha1, -alpha2, -beta1, -beta2, -gamma1 and -gamma2 were detected by quantitative PCR (Q-PCR) and immunohistochemical staining (IHC). Their expression levels in ovarian tumors were compared with normal controls and also correlated with clinicopathological parameters. RESULTS: Except AMPK-alpha1, expressions of the other five AMPK subunits are significantly higher in ovarian carcinomas as determined by Q-PCR. Although IHC detection of AMPK-gamma1 and -gamma2 were not successful, over-expressions of AMPK-alpha2, -beta1, and -beta2 were further confirmed by IHC. Over-expressions of various AMPK subunits occurred independently and were mainly detected in the cytoplasm. Interestingly, AMPK-alpha2 and -beta1 were also detected in the nucleus and cell membrane, respectively. Clinical correlation analyses indicate that expressions of different AMPK subunits are associated with different subtypes of carcinoma. High expression of AMPK-alpha2 is significantly associated with endometrioid carcinomas. On the other hand, high expressions of AMPK-beta and -gamma subunits are associated with mucinous and serous carcinomas, respectively. Furthermore, high expressions of AMPK-beta1 and -gamma2 are also associated with early and late stages of disease, respectively. Finally, patients with high expression of AMPK-alpha2 had better prognosis. CONCLUSIONS: Aberrant expressions of AMPK subunits may play important roles in ovarian carcinogenesis. Each AMPK subunit may have its own function other than just a component of the AMPK molecule. Correlations with clinical parameters suggest that expressions of AMPK subunits have different clinical implications in ovarian cancer development.