The Study Of The Function Of Hetl In Muscle Regeneration

HET1is a kind of guanine nucleotide exchange factor of Dbl domains protein family. It can regulate the conjunction state of protein G of Rho family with GTP or GDP. When G protein is connected with GTP, the activity of GTPase is activated and its downstream effector proteins are activated and transmitted the relevant cell signals. When G protein is combined with GDP, its GTPase is inactivated and it does not play a role in relevant activity. The binding state transition of Rac1, Cdc42and Rho A binding to GTP and GDP is controlled by HET1, which plays an important regulatory role. HET1is widely and highly expressed in brain, heart, muscle and other excitable tissues. It can affect the cell shape, division, migration, cell adhesion and cell polarity by activation of a small G protein. Previous studies showed that HET1was upregulated after muscle injury, and exogenous overexpression of HET1using adenovirus can accelerate the process of muscle repair after damage, while overexpression of HET1the activity of Racl、Cdc42and the GTPase of RhoA are increased. In vitro, using mouse skeletal muscle cell lines found that HET1was able to induce the differentiation of C2C12cells and inhibit their differentiation to adipocytes.. Nevertheless, due to lack of in vivo gene knockout mouse model, the function of Hetl in muscle regeneration and differentiation remains unclear in vivo. We first established HET1f1/f1mouse and crossed it with EII-Cre transgenic mouse to obtain HET1knock out mouse. PCR and Southern Blot analysis showed that the gene fragment of HET1was removed from knockout mice. But the life condition of HET1full knockout mice and wild-type mice showed no difference, and they both in good condition. The tissue sections of heart, muscle, stomach and intestine observed no significant difference between the two. When a small muscle injury occurs, the muscle can heal itself. We successfully induced a muscle injury model by injecting cardiotoxin (CTX) into mouse skeletal muscle. We injected50μl of10μg/ml cardiotoxin and checked the expression variation of c-myc, MyoD and myogenin in7days. The results showed that5days after injection, the proliferation of muscle satellite cell and differentiation of muscle cells reached to its peak. And we compared the tissue section of muscle injury part of wild type and HET1knockout mice after5days or10days of injection and found that the recover process of HET1knockout mouse was significantly delayed when compared with wild type. Our results are the first in vivo proof of the important function of HET1in regulating muscle injury repair. In addition, we designed an experiment and successfully separated the satellite cells of mouse skeletal muscle and induced them differentiate into skeletal muscle cells. And ultimately, they will form myotubes in vitro. Our study laid significant foundation for further study of the function of HET1in muscle regeneration.