| IntroductionCell is an essential unit of structure and function in organism,which is intercoordination in the process of metabolize. Apoptosis is a form of cell death and a fundamental biological phenomena,which partake not only the natural process of growing development and aging but also a principal element of the pathogenesis for a variety of diseases. The signal transduction mechanism of apoptosis is an important and fundamental problem in life science. The main signal transdution pathways in apoptosis are clear at present.Lysosome is a critical structures of cell apoptosis. There are different kinds of signal pathways in different kinds of apoptosis signal. The mechanisms of signal transduction for apoptosis between lysosome and mitochondria remains unclear. Our experiment is on the basis of previous research. We founded that K562 cell can underwent apoptosis after inducing by Glucosamine Sulfate(GS) and this is related to the release of Cathepsin D(Cat D) from lysosome , which can down-regulate Bcl-XL. But the relationship and modulation mechenisms of Cat D and Bcl-XL remain unclear. In order to find whether exist the molecular basis of the interactions between Cat D and Bcl-XL, we did the following reaearch.ObjectiveTo explore the molecular mechinisms between Cat D and Bcl-XL among the signal transduction in apoptosis of K562 cells induced by Glucosamine Sulfate. Apoptosis models and identification were made. And immunofluorescence,Western Blot and RNAi experiments were did.Methods1.The apoptosis model of K562 was made by 5.0 mmol.L-1 glucosamine sulfate. The methods of hematoxylin-eosin and Hoechst33342 dyeing stain, flow cytometry and DNA ladder were adopted to identify the apoptosis model.2.The methods of immunofluorescence,Western Blot were used to detect the mechanism of action between Cat D and Bcl-XL before apoptosis, after apoptosis and after inhibition for Pepstatin A.3.Cat D-specific siRNA and Bcl-XL specific siRNA (Santa Cruz) and one pair -of non-specific siRNA (NC) were used. The siRNAs were transfected into K562 cells with cationic liposome, then RT-PCR and Western Blot were used to detect mRNA and protein expression of Cat D and Bcl-XL.Results1.After induced by GS, the viability of K562 cells decreased following the increasing of concentration and time. The viability of K562 cells inducing by 5.0 mmol.L-1GS is obviously decreased. Compared with K562 cells of control group,the K562 cells induced by 5.0 mmol.L -1 GS show cells shrinkage, membrane unsmooth and blebs appearing. DNA ladders can also be seen clearly in the experiment group.2.The colocalized signals of Cat D and Bcl-XL were enhanced in K562 cells after apoptosis induced by 5.0 mmol.L -1 GS for 72h. The expression of Cat D was increased and Bcl-XL decreased. The colocalizd signals of Cat D and Bcl-XL were increased in the pepstatin A group in which the apoptosis of K562 cells were induced by 5.0 mmol.L-1GS for 72 h after inhibiting by Pepstatin A than GS induced group. In this group the expression of the Cat D was decreased but the expression of Bcl-XL protein wasn't changed.3.After transfected with siRNA of Cat D or Bcl-XL,the colocalized signals of Cat D and Bcl-XL were become weakened as the expression of Cat D or Bcl-XL were reduced. In the group of transfected by Cat D siRNA induced by 5.0 mmol.L-1GS for 72 h, the colocalized signals of Cat D was increased. The expression of protein of Cat D was increased and Bcl-XL was not obviously changed after induced by 5.0 mmol.L-1 GS for 72h.Conclusions1.The apoptosis model of chronic myelogenous leukemia K562 cells was established successfully. The viability of the cells in the apoptosis group was obviously decreased.2.The enhanced colocalization of Cat D and Bcl-XL in K562 cells induced by glucosamine sulfate suggested that the two molecules maybe exist some basis of interaction in apoptosis. After induced by 5.0 mmol.L-1glucosamine sulfate for 72 h,the signals of colocalization relationship between Cat D and Bcl-XL were enhanced as the expression of Cat D was increased while the expression of Bcl-XL was decreased. Induced by 5.0 mmol.L-1GS for 72 h after inhibiting by Pepstatin A,the colocalized signals of Cat D and Bcl-XL shows that the signals of Cat D were enhanced than GS induced group . The expression of Cat D was decreased while Bcl-XL had no change.3.The colocalized signals of Cat D and Bcl-XL were decreased when K562 cells were transfected with Cat D and Bcl-XL -specific siRNA. After transfered with Cat D siRNA, the signal of Cat D was reduced while the signal of Bcl-XL was comparatively increased. After transferred with Bcl-XL siRNA, the signal of Cat D was comparatively increased. After transfered with Cat D siRNA and Bcl-XL siRNA,the expression of Cat D and Bcl-XL were decreased. After Cat D siRNA induced by 5.0 mmol.L-1 GS for 72 h, the protein expression of Cat D was increased as the protein expression of Bcl-XL was not obviously alteration. The groups of Negative control showed there is no any kind of expression。...
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