| Objectives (1) To clone the gene encoding mouse 3 -defensin-6. (2) To investigate the effect of acute lung injury(ALI) on beta-defensin-4,6(mBD-4,6) gene expression in mouse lung, to discuss the role of mBD-4,6 in development of ALL (3) To study the expression and regulation of mBD-4,6 in milk mice and adult mice. (4) To develop a real-time quantitative PCR method for detection of expression of hBD-3 gene and investigate expression levels of hBD-3 gene in lung cancer and lung normal tissue.Methods (1) Total RNA was extracted from mouse lung tissue after LPS stimulation and complementary DNA was made by reverse transcription as template for polymerase chain reaction. PCR product of expected size was then inserted into pGEM-T-EASY cloning vector. Recombinant pGEM- Mbd-6 vector was transduced into competent cell JM109. PCR were performed to identify the clone containing the DNA. Fragment of interest, followed by sequencing with sequencer.(2) Healthy adult mice were randomly divided into two groups: control group and ALI group. In ALI group acute lung injury was induced with intra-peritoneal LPS.The control group was induced with intra-peritoneal the same dose NS. Lungs were harvested in different time after LPS stimulation or NS. Total RNA was extracted from pulmonary tissue mBD-4,6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR). DNA sequencing was used to confirm the specificity of mBD-4,6 cDNA fragment.(3) Two groups of animals: milk mice group and adult mice group. Two groups was induced with intra-peritoneal LPS. Lungs were harvested in different time after LPS stimulation or NS. Total RNA was extracted from pulmonary tissuemBD-4,6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction(RT-PCR).(4) Using SYBR Green I, with GAPDH as reference, a real-time quantitative PCR method was established. hBD-3 gene expression levels of 13 normal and 16 lung cancer tissue were detected and analyzed.Results (1) We obtained a 263bp DNA fragment that is identical to the mBD-6 cDNA sequence registered in GenBank. (2) Acute lung injury induced by intra- peritoneal LPS was confirmed by microscopic pathological examination. There were no significant mBD-4,6 mRNA expression in mouse puhnonary at different time intervals in control group and 6h in ALI group. In ALI group a marked increasing in puhnonary mBD-4,6 mRNA was found on 12h,ld,3d after LPS stimulation.(3) There were mBD-4,6 mRNA expression in both milk and adult mice groups after LPS stimulation 12 h. Real-time Fluorescent quantitative confirm that mBD-4,6 mRNA expression of milk group after LPS stimulation were significantly lower than adult group (P<0.01) . (4) Expressions of hBD-3 mRNA were significantly different in normal lung and lung cancer tissue, Expression of hBD-3 mRNA in lung cancer was significantly higher than in normal lung, the expression ratio of lung cancer to normal lung is 3.31.Conclusion (1) MBD-6 gene was successfully cloned from mouse lung tissue, providing opportunity to obtain genetic engineering product, to therapy clinical infection and further study action mechanism of mBD-6. (2) mBD -4,6 mRNA expressions show a up-regulative expression pattern. ALI can up-regulate the mBD-4,6 mRNA expression. The up-regulation of mBD-4,6 mRNA expression may be involved in the pathogenesis of ALI through participating in "cell network" and "cytokine network".(3) mBD -4,6 mRNA expressions show a up-regulative expression by LPS in both milk and adult mice lung tissue, their expression difference are related to age and auxo. These result reveals that when cell immune defence system of milk mice is not mature, their innate immunity...
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