The Application Research Of Atpase Staining In Cultured Skeletal Muscle Cells

Objective:Using myosin ATPase histochemical staining method to analysis and identify the rabbit skeletal muscle tissues and the subtypes of cultivated cells.Through comparison with the corresponding MHC gene expression level,to discuss the application feasibility of myosin ATPase histochemical staining method in identifying muscle cells and the relationship with muscle fiber type conversion process in vitro.Methods:1 Take the rabbit slow muscle fibers(Type I,inherent semi-membrane muscle subdivision)and fast muscle fibers(Type II,dorsal part of deputy semi-membrane muscle subdivision)to identify the type of muscle fibers via ATPase staining method.2 Take the rabbit slow muscle fiber and fast muscle fiber tissue blocks to separate,cultivate and observe primary cells.3 Making cell-attached slides after the two subtype muscle fibers growing well,and stain the cell-attached slides via ATPase histochemical staining after cell membrane rupture of slides on Day 3 and Day 7.Then,observing and identifying muscle fiber types,and comparing with MHC isotype detection.The MHC gene isotype of the two type muscle cells can be detected by RT-PCR.Results:(1)The result of ATPase histochemical stain of rabbit skeletal muscle fibers shows that after the acid preincubation process in pH4.3,the Type I skeletal muscle cells(i.e.,slow muscle fiber)are dyed in deep color,while the Type II muscle fibers(i.e.,fast muscle fiber)are dyed relatively shallow.Conversely,after the alkali preincubation process in pH10.4,the Type II fibers are stained deeper in deep color,while the Type I fibers are shallow.(2)Separate and cultivate the primary cells to form them into myoblast system with the density controlled in of 1×10~5/ml for subsequent experiments.(3)In the cell-attached slides of Day 3,after the acid preincubation process in pH4.3,the proportion of the cells stained deeper in the original Type I skeletal muscle fibers was decreased with some lighter dyed cells appeared,while the original Type II fibers are all in shallow staining.Whereas,after the alkali preincubation process in pH10.4,the original Type II fibers are nearly all in deep color,while some deep dyed cells occurred in the slides of original Type I fibers.In the cell-attached slides of Day 7,after the acid preincubation process in pH4.3,the proportion of the cells stained deeper in the original Type I skeletal muscle fibers has a further reduction with the lighter dyed cells much more,while the original Type II fibers are still all in shallow staining.Whereas,after the alkali preincubation process in pH10.4,the original Type II fibers are still nearly all in deep color,while the number of deep dyed cells grow continually in the slides of original Type I fibers with the percentage of shallow stained cells decreased.Compared with MHC expression,there was a decrease in MHC I expression of the original slow muscle fibers in Day 3,while the MHC II expressed clearly.In Day 7,the expression of MHC I continually dropped with MHC II increased obviously.However,the original fast muscle fibers both in Day 3 and Day 7 were still expressing the MHC II type.Conclusion:1 ATPase histochemical staining can show the types of cultured skeletal muscle cells.It can be used to provide morphological basis for the study of fiber type conversion.2 By means of comparing with the result of MHC gene isotypes expression level,we can verify that the transformation between skeletal muscle fiber types existed during cultivation process in vitro.