| Part ? CDDP inhibits proliferation of rats' astrocytesObjectiveAs the most abundant cell type in CNS,defects of astrocytes function have been implicated in a variety of diseases.Cisplatin(CDDP)is a typical kind of chemotherapeutic drugs that is widely used to treat numbers of cancers but leads to neurocognitive impairment in patients,but little is still known about the damage effects of chemotherapeutic drugs on astrocytes.The purpose of the experiment in this part is to explore the effects of CDDP in astrocytes' proliferation.Methods1.The rats' astrocytes were cultured in vitro;2.All cells were cultured in 96-well plates at a density of 2×103 per well,then 6.25?M,12.5 ?M,25 ?M,50 ?M,100 ?M CDDP were added into cells for 24h to detect cells viability;3.Discarding the culture medium containing CDDP in 96-well plates,then the fresh culture medium without CDDP were added into 96-well plates for 96 h,120 h,144 h.The cells viability was detected;4.All cells were cultured in 24-well plates at a density of 2×104 per well,then 6.25?M,12.5 ?M,25?M,50?M,100 ?M CDDP were added into cells for 24h to detect the proportion of Ki67 positive cells;5.Discarding the culture medium containing CDDP in 24-well plates,then the fresh culture medium without CDDP were added into 24-well plates for 96 h to detect the proportion of Ki67 positive cells.Results1.The purified astrocytes could express the specific marker GFAP;2.After treatment with 12.5 ?M,25 ?M,50 ?M,100 ?M CDDP for 24 h,the cells viability decreased;after treatment of 6.25 ?M CDDP for 24 h,the cells viability was not decreased;3.After treatment with 12.5 ?M,25 ?M,50 ?M,100 ?M CDDP for 24 h,the proportion of Ki67 positive cells decreased;after treatment of 6.25 ?M CDDP for 24 h,the proportion of Ki67 positive cells was not decreased;4.After recovery in CDDP-free medium to 96 h,120 h,144 h,the cells viability was still decreased in 12.5 ?M CDDP-treated cells;5.After recovery in CDDP-free medium to 96 h,the proportion of Ki67 positive cells was decreased in 12.5 ?M CDDP-treated cells than that in control cells.Part ? CDDP inhibits proliferation of rats astrocytesObjectiveIn the first part,the low-dose CDDP could inhibits cells viability and decreased the ability of proliferation.The purpose of the experiment in this part is to explore the effects of CDDP in astrocytes' apoptosis.Methods1.The rats' astrocytes were cultured in vitro;2.All cells were cultured in 24-well plates at a density of 2×10 per well,then 6.25?M,12.5 ?M,25 ?M,50 ?M,100 ?M CDDP were added into cells for 24h to detect apoptotic cells by using TUNEL method;3.All cells were cultured in 24-well plates at a density of 2×104 per well,then 6.25?M,12.5 ?M,25 ?M,50 100 ?M CDDP were added into cells for 24h;then all cells were cultured in CDDP-free medium to another 96 h,120 h,144 h,then all apoptotic cells were detected by using TUNEL method;4.All cells were cultured in 6-well plates at a density of 2×105 per well,then 6.25?M,12.5 ?M CDDP were added into cells for 24h;then all cells were cultured in CDDP-free medium to 96 h,120 h,144 h,then all apoptotic cells were detected by using flow cytometry(FCM).Results1.After treatment with 25?M,50 ?M,100 ?M CDDP for 24 h,the proportion of TUNEL positive cells increased;2.After treatment with 6.25 ?M,12.5 CDDP for 24 h,the proportion of TUNEL positive cells did not increase;3.All cells were cultured in CDDP-free medium,the TUNEL-positive cells number was still increased in 12.5 ?M CDDP-treated cells than that in control group;4.All cells were cultured in CDDP-free medium,the apoptotic cells number was increased in 12.5 ?M CDDP-treated cells than that in control group cells.Part? CDDP inhibits cellular autophagy of rats astrocytesObjectiveIn previous experiment,low-dose CDDP inhibited astrocytes proliferation and induced apoptosis.The purpose of the experiment in this part is to explore the effects of CDDP in astrocytes' autophagy.Methods1.The rats' astrocytes were cultured in vitro;2.After treatment with CDDP in purified astrocytes,the total protein was extracted,and autophagic proteins LC3-?,ATG5,ATG7,P62 was detected by using Western blot;3.The purified astrocytes were seeded at a density of 2×104 in 24-well plates,then the Ad-mRFP-GFP-LC3 was added into the cells and the MOI(Multiplicity of infection)was 200;4.After treatment with CDDP,the RFP+/GFP+yellow puncta or RFP+/GFP-red puncta was detected in Ad-mRFP-GFP-LC3 transfection astrocytes;5.The autophagy inhibitor chloroquine diphosphate(CQ)was used to inhibit the autophagic flux,then autophagic proteins was detected;6.After treatment with CDDP,the supernatant protein was prepared,the GSR-CAA-67 protein chip was used to detect the expression;7.The purified astrocytes were seeded at a density of 2×105 in 6-well plates,the lentivirus LV-NRP2 and LV-NC was added into the cultured cells with the MOI=200.Then all cells were cultured with CDDP,the Western blot was used to detect the expression of autophagic protein LC3-?.Results1.After treatment with 12.5?M,50 ?M,100 ?M CDDP for 24 h,the autophagic protein LC3-?,SQSTM1/P62,ATG5,ATG7 was decreased;2.After treatment with CDDP in Ad-mRFP-GFP-LC3 transfection cells for 24 h,the RFP+/GFP+yellow puncta(autophagosome)was decreased in CDDP-treated cells,and the RFP+/GFP-red puncta(autolysosome)was decreased in CDDP-treated cells;3.All cells were cultured in CDDP-free medium to 96 h,the number of yellow and red puncta still decreased than the cells in control group;4.After treatment with CQ and CDDP for 24 h,the expression of LC3-? in CQ+CDDP group was less than that in CQ group;5.After treatment with CQ and CDDP for 24 h,all cells were cultured in CDDP-free medium for another 96 h,the expression of LC3-? in CQ+CDDP group was still less than that in CQ group;6.After treatment with CDDP for 24 h,all cells were cultured in CDDP-free medium for another 96 h,120 h,144 h,the expression of LC3-? was decreased than that in control group;7.After treatment with CDDP for 24 h,all cells were cultured in CDDP-free medium for another 96 h,120 h,144 h,the expression of ATG5?ATG7 upregulated into the normal level,while the expression of SQSTM1/P62 was higher than that in control group;8.The protein was detected in 12.5 ?M CDDP-treated cells in GSR-CAA-67 protein chip,the results showed that Neuropilin-2(NRP2),GAS-1,Notch-2 were all decreased,especially NRP2;9.After treatment of LV-NRP2,the expression of NRP2 was upregulated in cells supernatant by using ELISA Kit;10.After treatment of LV-NRP2,the expression of LC3-? was increased than that in control group.Moreover,the expression of LC3-? in LV-NRP2+CDDP was higher than that in LV-NC+CDDP.Conclusion1.Low-dose CDDP inhibited the cells viability and proliferation of astrocytes;2.Low-dose CDDP induced apoptosis of astrocytes;3.Low-dose CDDP inhibited the cellular autophagy.And the CDDP-caused autophagy dysfunction was attributed to the downregulation of autophagic proteins;4.Low-dose CDDP decreased the expression of NRP2;after recovery of NRP2,the overexpression of NRP2 protected against CDDP-caused the autophagy dysfunction;5.CDDP could inhibit cells proliferation,induce apoptosis,and cause autophagy dysfunction as well.And CDDP-caused astrocytes damage might be related to the CDDP-related nervous system impairment. |
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