筛选细菌致病基因的几种常用方法

以致病基因作为靶点的药物为我们提供了从基因水平预防、治疗疾病的可能,筛选致病基因,成为攻克细菌、病毒乃至遗传因素导致的疾病的一个重要课题。筛选方法不断地改革与创新,针对不同的研究目的需要合理地选择筛选方法,本文简要介绍几种常用的细菌致病基因的筛选方法。     

Somatostatin receptor 1–5; expression profiles during rat development

Background

Somatostatin acts through five receptor subtypes (SSTRs 1–5). We aimed to investigate SSTRs mRNA expression and protein distribution in whole rat embryos, with special emphasis on the pancreas.

Material and methods

Rat embryos were collected on embryonal days 10, 11, 12, 14, 15, 17, 19, 21, and at birth. Presence of SSTRs was investigated with RT-PCR techniques and immunohistochemistry.

Results

There was no SSTR5 mRNA expression in the whole rat embryos. All SSTR1–5 proteins were observed at embryonal day 10, but the localization varied between the different subtypes. From day 11 to birth SSTRs protein presence increased with time in major structures such as skin and cartilage. It remained similar over time in the heart and liver. In the fetal pancreas mRNA expression of SSTR2 and 4 was detected at day 14, and there was an increase up to birth. Only SSTR1 protein co-localized to a higher extent with the islet hormones studied. SSTR2 was present in all islet endocrine cells except for β-cells. In contrast, the immunostaining for SSTR3–4 was co-localized with insulin and PP, and, finally, SSTR5 with glucagon and pancreatic polypeptide. In mRNA isolated from whole rat embryos SSTR1-2 and SSTR4 expression showed a peak at day 14, while SSTR3 mRNA was not present until day 15.

Conclusion

The present data suggest a role for SSTRs during the development of the rat embryo. Subsequent functional studies may elucidate regulatory roles of specific SSTRs for the growth and differentiation of the pancreas as well as other organs.     

腹腔灌洗液中CK20m RNA水平与结直肠癌不同术式的研究

目的探讨腹腔灌洗液中CK20m RNA的临床意义以及腹腔镜直肠癌根治术的临床应用价值。方法选择49例直肠癌手术患者,利用RT-PCR技术分别检测以上患者腹腔灌洗液中CK20m RNA表达,从而探究CK20m RNA的临床价值以及评价腹腔镜直肠癌根治术的临床实用性。结果 10例良性病变腹部手术组患者腹腔灌洗液中CK20m RNA无明显表达。39例结直肠癌患者腹腔灌洗液中可见CK20m RNA表达,术前、术中以及术后的总表达率为40.2%(47/117),且术前、术中以及术后CK20m RNA的表达率比较差异无显著性(P0.05)。17例开腹手术组和22例腹腔手术组术前、术中以及术后CK20m RNA阳性表达率的比较差异无显著性(P0.05)。结论腹腔镜结直肠癌根治术治疗结直肠癌安全有效,与开腹手术相比并不增加癌细胞局部种植的风险。     

Localization of apoptotic and proliferating cells and mRNA expression of caspases and Bcl-2 in gonads of chicken embryos

The aim of the present study was to analyze participation of apoptosis and proliferation in gonadal development in the chicken embryo by: (1) localization of apoptotic (TUNEL) and proliferating (PCNA immunoassay) cells in male and female gonads and (2) examination of mRNA expression (RT-PCR) of caspase-3, caspase-6 and Bcl-2 in the ovary and testis during the second half of embryogenesis and in newly hatched chickens. Apoptotic cells were found in gonads of both sexes. At E18 the percentage of apoptotic cells (the apoptotic index, AI) in the ovarian medulla and the testis was lower (p < 0.05) than in the ovarian cortex. In the ovarian medulla, the AI at E18 was lower (p < 0.05) than on E12. In the testis, the AI was significantly lower (p < 0.05) at E18 than at E15 and 1D. The percentage of proliferating cells (the proliferation index: PI) within the ovary significantly increased from E15 to 1D in the cortex, while proliferating cells in the medulla were detected only at E15. In the testis, the PI gradually increased from E12 to 1D. The mRNA expression of caspase-3 and -6 as well as Bcl-2 was detected in male and female gonads at days 12 (E12), 15 (E15) and 18 (E18) of embryogenesis and the day after hatching (1D). The expression of all analyzed genes on E12 was significantly higher (p < 0.05) in female than in male gonads. This difference was also observed at E15 and E18, but only for the caspase-6. The results obtained showed tissue- and sex-dependent differences in the number of apoptotic and proliferating cells as well as mRNA expression of caspase-3, -6 and Bcl-2 genes in the gonads of chicken embryos. Significant increase in the number of proliferating cells in the ovarian cortex and lack of these cells in the ovarian medulla (stages E12, E18, 1D) simultaneous with decrease in the intensity of apoptosis only in the medulla indicates that proliferation is the dominant process involved in the cortical development, which constitutes the majority of the functional structure of the fully developed ovary. No pronounced changes in the expression of apoptosis-related genes found during embryogenesis suggest that they cannot be considered as important indicators of gonad development. The molecular mechanisms of the regulation of balance between apoptosis and proliferation in developing avian gonads need to be further investigated.