| Macrophage is the most important cell type in human immune system when counter Mycobacterium tuberculosis(Mtb). It has been demonstrated that macrophage is closely related with the whole infection process of Mtb by a large amount of experimental results during these years. The process includes recognition of Mtb, control and killing them. However, some of the recent researches show macrophage may become the harbour for Mtb under the interference by them. Even more, macrophage can provide Mtb with space and energy sources and help them to disseminate to other places of body as a kind of carrier. Because of the two contradictory views, we choose macrophage as our research object. We want to compare the difference of gene expression profile of macrophage in normal lung tissue and diseased lung tissue of phthisis in order to identify the genes which play crucial role in the process of Mtb infection. As 90% persons don't transform into patients after they infecte Mtb and only 10% infected persons may develop into tuberculosis at some time point of their whole life. So we want to find the genes related to the happening of tuberculosis through this experiment. On the other hand, most of the pulmonary tuberculosis patients just need medicine treatments. Only very few of them must accept surgical operations for their serious pathological changes such as tuberculous thick walled cavity, tuberculoma and damaged lung. Therefor, we hope we can find some genes which are associated with the formation of these serious lesions by doing this research. After this, we might work out some methods to interfere the process of the disease progression according to these related genes, so as the morbidity of the serious diseases may decline. Under the help of the results, we can effectively control the incidence of usual pulmonary tuberculosis and the severe forms of it without cutting off the transmission route.However, after sereval times of sample processing, we found there were many difficulties in sorting macrophages from lung tissues. First, different patients have various course of disease, use various kind of medicine. So, the lung tissues getting from patients have different conditions. Second, we lack of standard method for sorting specific type cells from human lung tissues. Therefor, we are not able to get single cell suspension of macrophage at present. It made us changing our experimental stratege. We directly use normal and diseased lung tissues for gene expression profile research.We find there are huge differential expression between the two types of lung tissues after analyzing the gene expression profile dates. When compared to diseased tissues, there are 332 genes up regulated and 102 genes down regulated to 2 fold in normal lung tissues. If we make the selecting criteria rising to 4 fold, the number of up regulation and down regulation genes will decrease to 19 and 2. The two down regulation genes are FER1L4 and PPIL5. And during the up regulation group, the AREG gene is very special. The up regulation fold of this gene in our three parallel experiments are more than 11 respectively. Beyond this, we also detected the expression level of some genes which are related with TNF as there have been lots of results can prove the close relationship between the cytokine TNF and granuloma formation and dissolution. The result was four TNF related genes were obviously up regulated in normal lung tissues compared to diseased ones. To sum up , we think the differential expression of all up and down regulated genes, especially PPIL5, FER1L4 and TNF genes result in the happening of lung tuberculosis and the formation of these serious tuberculosis lesions.In addition, we compared the expression level of Peripheral Blood Mononuclear Cell(PBMC)before surgery with the one after surgery. Through density gradient centrifugation, we isolated PBMC from blood samples of pulmonary tuberculosis patients in order to do the gene expression profile research. The purposes of this experiment was to understand the change of patients'immune state after wiping out mycobacterium tuberculosis burden by surgery. The results demonstrated that there were 38 genes up regulated and 91 genes down regulated expression to 2 folds. When rising the selecting criteria to 4 fold, we can not find any gene which differential expressed to this in our three parallel experiments. So, we made 3 folds as selecting criteria. The results showed that there were only 6 genes down regulated expression and 2 genes up regulated expression in preoperation PBMC. After checking the function of these differential expression genes, we found there were not obvious relationships between them and the immune response aimed at mycobacterium tuberculosis. It made us think that the systemic immune response, represented by PBMC, might not play the critical effect in controlling the mycobacterium tuberculosis infection. We did another experiment about genotyping. We use small size samples of Mtb getting from extrapulmonary tuberculosis and pulmonary tuberculosis for genotyping research in order to understand the differences of genotype between them. Through it , we can accumulate some dates and materials about the relationship of extrapulmonary tuberculosis and pulmonary tuberculosis. still further, it will help to offer relevance proof for working out the distinct drug using standards of individualized therapy for the patients who are suffering the extrapulmonary tuberculosis or pulmonary tuberculosis.Totally 29 Mtb isolated strains were typed by Spoligotyping and VNTR genotyping, including H37Rv,14 isolated strains collected from extrapulmonary tuberculosis patients(E-Mtb) and 14 isolates from pulmonary tuberculosis patients(P-Mtb).Comparing these results, 14 P-Mtb isolates can be divided into 5 distinct patterns by Spoligotyping(9 beijing genotype,2 T2 genotype and three other genotypes) and 14 patterns by VNTR. In contrast, 14 E-Mtb isolated strains are grouped into the completely same cluster assessed by both motheds. The cluster can be shown as a column of figures 777777777777771 by Spoligotyping and 013582202252 by 12 VNTR locus we used.There are significant differences in genotype of Mtb between extrapulmonary tuberculosis patients and pulmonary tuberculosis ones. Of P-Mtb, Beijing genotype is the main epidemic strains. In this experiment, it holds about the equal proportion about 65% of all P-Mtb with previously reports. But in E-Mtb, there are not any isolated strains were Beijing genotype. Furthermore, all of E-Mtb possess the same genotype. So, we think that this genotype may action as the main epidemic strains of extrapulmonary tuberculosis, and it can be able to invasion extrapulmonary tissue much easier than other strains. |
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