| Cancer is still the leading cause of human death. Radiation and chemotherapy are the most used therapies to treat cancer. However, they usually bring about the hematopoietic and immune suppression, which are the most serious and life threatening side-effects, of the patient. Treating and managing these side-effects has become the active field for cancer therapy since the introduction of radiochemotherapy.In order to induce bone marrow damage and regeneration, we use 5-fluorouracil (5-Fu) to inject mouse. The result shows that, after 5-Fu injection, the peripheral blood and bone marrow cell numbers of mouse begin to decrease. On the 7th day after treatment, the numbers reach the lowest level, and then start to increase gradually. Within 2-3 weeks, the damaged bone marrow is fully recovered. It’s the same to human after chemotherapy. We hypothesized that a series of regulatory genes determine the speed of bone marrow regeneration.Using gene expression microchips, we analyzed the expression pattern of bone marrow genes using the total mRNA obtained from mouse bone marrow after 5-Fu injection. 3,9000 genes were identified with expression pattern fit with our hypothesis. Resistin is one of them.Resistin, a cysteine-rich polypeptide, is a recently discovered adipocytokine and named for its role of insulin resistant. Resistin is mainly expressed in adipocyte of rodent. Three dimensional structure of Resistin has been resolved. Resistin represents an important molecular linker between type II diabetes and obesity through affecting liver glycogen metabolism. It also induces angiogenesis through regulation of microvascular endothelial cells and smooth muscle cells. Furthermore, Resistin reduces the size of myocardial infarction in animal models. In our gene expression microchips of bone marrow, the expression pattern of resistin after chemotherapy shows remarkable feature of first increasing and then declining during bone marrow regeneration, which suggests its protection of the bone marrow from chemotherapy damage or relation to the recovery of the damaged bone marrow. The purpose of this project is to produce murine resistin recombinant protein (rmResistin) and to investigate its regulatory effects on hematopoiesis and its possible therapeutic value in treatment of chemotherapy induced bone marrow suppression.I cloned the murine resistin (mResistin) full length cDNA, and constructed the eukaryotic expression plasmid phCMV1-mResistin. The plasmid was introduced into the tibia muscle of mouse by electroporation. To further study the role of resistin in protection of bone marrow from chemotherapy damage, we constructed the coding sequence of mature mResistin into the prokaryotic expression plasmid pET-28a. The plasmid was induced to produce the recombinant murine resistin protein (rmResistin) in E. coli BL21(DE3). The murine resistin is a low molecular protein of 10.4 kDa with no glycosylation. After denaturation and renaturation, the recombinant protein was purified by ion-change chromatography (S Sepharose) and identified by western blot. The biologic activity and endotoxin level of this recombinant protein were measured. Then I injected this protein into normal mice to study its regulatory role in bone marrow regeneration.The main methods and results of our work are as follows: First, we got the mResistin gene fragment from murine bone marrow cDNA library, which was obtained by RT from the totall RNA of murine bone marrow. After agarose gel electrophoresis, we saw the 345 base pairs gene fragment. The gene fragment was cloned into phCMV1 to construct recombinant plasmid, which was transformed into the cloning bacteria E. coli DH5α. PCR analysis was used to select the right clone. The sequencing result showed that the fragment had 2 mutants, which were repaired by 2-step PCR. Then the plasmid was reconstructed and introduced into the tibia muscle of mouse by electroporation. The results showed that not only the numbers of WBC and BMC, but also the percentage of the S cycle of BMC, increased compared with the control group. To further study the role of resistin in protection of bone marrow from chemotherapy damage, we got the coding sequence of mResistin using phCMV1-mResistin as PCR template. The 285 base pairs gene fragment was cloned into pET28a(+) to construct prokaryotic recombinant expression plasmid pET-28a-mResistn, which was transformed into the expressing bacteria E. coli BL21 (DE3). Restriction enzyme analysis was used to select the right clone. The sequencing result showed that it was indentical to mResistin gene encoding the mature protein. The protein expressing condition was optimized, which is induced for 8 hours in 42℃, the LB liquid culture medium contained 100μg/ml Kana, and IPTG concentration was 1 mmol/L. After induced by IPTG, the protein was found mainly in inclusion bodies, and its molecular weight is 10.4 kDa. The bacteria were centrifuged, sonicated and washed for three times. We used 6 M guanidine hydrochloride to denature the inclusion bodies, then the proteins were renatured with renature solution containing GSH/GSSH (pH 6.0). At last, the protein solution was obtained after ion exchange chromatogram. The protein concentration was 0.548 mg/ml measured by BSA protein quantify method, and the protein purity was more than 95% analyzed by SDS-PAGE. The endotoxin and biologic activity of this recombinant protein were also measured. The endotoxin content was less than 1 EU/μg by endotoxin measure kit. The protein can induce hematopoietic progenitor/stem cells to proliferate and differentiate. We injected the protein into normal mice to study their effects on bone marrow regeneration. It showed that rmResistin significantly increased the number of bone marrow cells. Pretreatment of mice with rmResistin before 5-Fu injection led to increased mortality, which indicated the activation of bone marrow hematopoiesis by rmResistin. Injection of rmResistin after 5-Fu treatment decreased the mortality, suggesting that the exogenous rmResistin played a positive role in the repair of bone marrow damage. The results of this study have laid foundation by providing recombinant proteins and functional analysis for the future study of regulatory roles of resistin during bone marrow regeneration. |
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