| Insulin like growth factor-1 (hIGF-1) is a kind of peptide which promotes differentiation and proliferation of various cells. Human IGF-1 shows a remarkable amino acid sequence homology to insulin, so some of actions like insulin. According to its action of decreasing glucose, IGF-1 had been supposed to cure diabetes mellitus (DM) from a long time ago. It proved that IGF-1 could help insulin to improve the metabolism of DM patients and reduce the dangerous caused by over insulin injection. When given the different dosage of IGF-1, the requirment to insulin decreases, HbAlc becomes low, and the abnormality of GH/IGF axis is improved, which is good to prevent and improve proliferate complications. In addition, the quantity of T lymphocytes decreases and the activity weakens so that many B cells defect, which results in I type DM (insulin dependent DM, IDDM). But after given IGF-1, CD4+ and CD8+ cells increase, the actions of CD4+ are inhibited, B cells are protected, all of these cause diabetic immuno-abnormality to be improved. II type DM (non-insulin dependent DM, NIDDM) is characterized by insulin resistance and weaken sensitivity to insulin, which are also critical factors of affecting the curative efficiency. IGF-1 can lower glucose through its own receptor, increase the sensitivity to insulin, reduce serum insulin, and improve the abnormality of glucose- resistance. In addition, IGF-1 plays the roles of reducing the blood adipose, PAI-1 and glycogen, in this way, IGF-1 may decrease the heart vessel and other vessal complications.As a factor of reducing glucose, IGF-1 can be used to IDDM and NIDDM patients, especially to some patients who have insulin resistance. At present, in clinical, recombinant hIGF-1, as a kind of assistant drugs,has been used to cure DM, diabetic complications, insulin-resistant syndromes, neural system diseases, kidney failure, and so on. The results are satisfied. However, injected IGF-1 cannot satisfy the normal physiological requirment and some side effects happen, such as muscle and joint pain, flush, edema, head pain and tired as well. Long time injection makes pain and uncomfortable to the patients. For these reasons, the research is aim to clone human IGF-1 gene, construct expression vector, transfect cos7 cells to detect the transcription, translation and protein expression in vitro by in situ hybridization, immunohistochemistry, and western blot, then examine the activity of hIGF-1 protein. At the same time, in order to get long time stable expression in vivo, AAV/hIGF-1 is reconstructed, marrow stromal cells (MSCs) are infected by high-titer viral particles packaged by HEK293 cells, then transplanted into DM mice model, the actions to DM and diabetic complications are observed. The results are as follows:1. Cloning hIGF-1 gene and constructing the expression vector pCI-neo/hIGF-1Total RNA was extracted from human fetal liver, and 710bp positive DNA band was obtained by RT-PCR method. The fragment cDNA was then cloned into the pUCM-T vector and the insert was sequenced after analysis of the recombinant pUCM-T plasmid by restriction enzymes. This sequence was proved to be correct compared with those in GeneBank　(M29644.1) and the eukaryotic expression plasmid(pCI-neo/hIGF-1) was then constructed.2. Labeling and detecting hIGF-1 cRNA probeThe plasmid pGEM-3zf/hIGF-1 was constructed by subcloning from pUCM-T/hIGF-1 and pGEM-3zf has T7 and SP6 promoter in order to prepare a digoxingen-labeled cRNA probe. The sense and antisense templates were purified by glassmilk after the plasmid was digested by EcoR I and Xba I restriction enzymes. The cRNA probes were identified with dot hybridization. The result of dot blot was shown that the positivereaction sites were purple-blue, and their densities were well-matched with the concentration and dilutions. This probe might provide a tool for identifying the gene of IGF-1 for research in different fields.3. Testing expression and activity of hIGF-1 geneTo examine the expression of IGF-1, COS-7 cells... |
