| Part Ⅰ Effect of hypoxia on 786-O cell proliferation, apoptosis,migration and invasiveness Objective:To investigate the proliferation, apoptosis, migration and invasion of renal cell carcinoma 786-O cells cultured under hypoxic conditions. Methods:Using containing penicillin and streptomycin and 10% fetal bovine serum 1640 culture medium cultured renal carcinoma 786-o cells, the experimental group cultured in the hypoxic incubator, anoxic conditions was 37 ℃, 5% CO2, 2% O2, 93% N2. Cells were cultured 8 hours, 12 hours and 24 hours, then the proliferation, apoptosis, migration and invasioness were detected. The control group 786-O cells were cultured under normal oxygen condition(37 C, 5%CO2, O2 95%). Using the MTT to detect renal cell carcinoma 786-o cell proliferational changes; By Hoechst staining method, under a fluorescent microscope to take five high-power field of apoptotic cells number was counted and flow cytometry was used to detected the apoptosis rate; By Transwell chamber, the cell migration ability was detected; By Transwell chambers with Matrigel coated, the invasive ability of cells was detected. The results were analyzed by SPSS19.0 for the single factor analysis of variance, P < 0.05 indicated that the difference was statistically significant.Results: 1. MTT colorimetric method: after renal cell carcinoma 786-o cells were cultured for 8 hours, 12 hours and 24 hours, the results of proliferation were follows ±respectively(hypoxia v.s normoxia): 97.6±3.6 v.s 96.5±4.2(8h); 95.4±2.5 v.s 96.7±2.5(12h), 98.1±2.3 v.s 97.3±2.6(24h). The results showed that there was no significant effect of hypoxia on the proliferation of 786-O cells at different time points(P > 0.05);2. Hoechst staining results: renal cell carcinoma 786-o cells were cultured for 8 hours, 12 hours and 24 hours and were apoptosis number were counted respectively(hypoxia v.s normoxia): 17.2±3.2 v.s 28.2±2.4(8h); 18.4±1.9 v.s 26.7±2.2(12h); 17.4±2.3 v.s 26.9±2.8(24h), statistical analysis found that under hypoxia condition apoptosis of 786-o cells is inhibited(P < 0.05).3. Flow cytometry results: renal cell carcinoma 786-o cells were cultured for 8 hours, 12 hours and 24 hours, the apoptosis rate(%)(hypoxia v.s normoxia) were follows: 5.2±1.2 v.s 11.2±1.9(8h); 6.7±1.1 v.s 9.7±1.2(12h); 6.5±0.9 v.s 9.9±1.6(24h). Results of statistical analysis at each culture time point indicates that under hypoxic conditions 786-o cells apoptosis rate was decreased(P < 0.05).4. Cell migration assay showed that after renal carcinoma 786-o cells were cultured for 8 hours, 12 hours and 24 hours, cell number counted as follows:(hypoxia v.s normoxia) 96±7.4 v.s 65±6.7(8h); 156±8.4 v.s 94±8.4(12h); 320±16.2 v.s 215±12.3(24h). At different time points, the number of cell migration in hypoxia group was significantly increased(P < 0.05).5. Invasion assay:(hypoxia v.s normoxia): 46±3.4 v.s 22±2.7(8h); 86±2.4 v.s 51±3.1(12h); 226±16.2 v.s 176±11.9(24h). After statistical analysis, the number of invasive cells in hypoxia group increased significantly(P < 0.05) was found in each culture time point. When the cells were cultured to 24 h under anoxic conditions, the invasion and migration of 786-O cells were detected, and the invsiving number was too much, so the hypoxia time of follow-up experiments was designed as 12 h.Conclusion:1. Hypoxia had no significant effect on the proliferation of 786-O cells.2. Hypoxia increases the anti apoptosis ability of 786-O cells.3. Hypoxia promotes the migration of 786-O cells.4. Hypoxia promotes 786-O cells invasion.Part Ⅱ Effects of hypoxia and NS398 on the expression of HIF 2 α, COX2 and E-cadherin/Snail in renal cell carcinoma 786-O cells Objective:To investigate the effect of hypoxia and NS398 on RCC 786-o cells protein expression related to hypoxia and invasioness. Evaluated effect of the specific inhibitor of NS398 on the expression of protein related to hypoxia and invasioness in renal cell carcinoma cell line 786-O. Methods:1. Synthetic si-RNA-HIF 1 α and si-RNA-HIF 2 α alpha were Purchased form Ruibo Company, the sequence were as follows: HIF 1αsense sequence(5 ’- 3’): GGGUAAAGAACAAAACACA; antisense sequences: UGUGUUUUGUUCUUUACCC(si RNA ID # 42840); HIF 2α sense sequence(5 ’- 3’) GGUUUUGUUGCUAGCCCUU; antisense sequences: AAGGGCUAGCAACAAAACC(si RNA ID No. 106447). In accordance with the instructions si-RNA-HIFs and Lipofectamine TM 2000 were mixed, using transient transfection method, the concentration of si-RNA-HIF 1α /2α were 20 n M, 50 n M and 100 n M respectively. After 48 hours, the cells were observed by fluorescence microscope to detect transfectional efficiency.2. Renal cell carcinoma 786-o cells transfected with si-RNA-HIF 1αand si-RNA-HIF 2α were cultured under hypoxia or normoxia, control group were renal cell carcinoma 786-o cells without transfection. Western blotting was used to detect intracellular HIF 1 α and HIF 2 α protein expressional changes in the transfected renal cell carcinoma 786-o cells after cultured for 3h, 8h, 12 h and 24 h repectivly. Transwell were used to detected invasioness. Control group were renal cell carcinoma 786-o cells without transfection.3. Renal cell carcinoma 786-O cells were divided into 2 groups: hypoxia +si-RNA-HIF 1α group, hypoxia+si-RNA-HIF 2α. Western Blotting was used to detect the expression of COX2 and E-cadherin/Snail protein in renal cell carcinoma cell line 786-O under hypoxic condition or nomoxia.4. Under nomoxia conditions, 20 u M, 40 u M, 80 u M end concentration of NS398 respectively and 786-O cells were co-cultured for 12 h, 24 h and 36 h. MTT was used to detect the cell proliferation activity, but in the control group, The concentration of NS398 is 0u M. Than the concentration of NS398 in 786-o cells without cell toxicity is screened.5. Renal cell carcinoma 786-O cells were divided into 4 groups, the normal oxygen group, hypoxia group, normal oxygen group+NS398, hypoxia+NS398 group, the incubation time is 12 h. The specific inhibitor of the epoxy synthase inhibitor NS398 concentration was 40 u M. The invasion ability of 786-O cells was detected by Transwell. Blotting Western was used to detect the expression of HIF 2α, COX2 and E-cadherin/Snail protein in renal cell carcinoma 786-O cells. Results:1. The transfection efficiency was calculated after the cells were counted under fluorescence microscope. The results showed(%): si-RNA-HIF1 α :15 ± 3.6(20n M); 46 ± 3.2(50n M); 74 ± 8.9(100n M). Si-RNA-HIF2α : 14.1±2.1(20n M); 36.2±5.3(50n M); 66±7.4(100n M). The results showed that si-RNA-HIFs was successfully transfected into renal cancer cells of 786-O at concertration of 100 n M.2. Western blot analysis showed that the RCC 786-O cell protein expression results(hypoxia v.s normoxia): 0.61±0.08 v.s 0.14±0.04; HIF2α 0.63±0.13 v.s 0.16±0.06. Single factor analysis of variance showed: P = 0.01 < 0.05. Hypoxia induced elevation of HIF1α and HIF2α expression in 786-o cells. However, when transfected with si-RNA-HIFs, the expression of target protein could not be detected in 786-O cells of renal cell carcinoma cells cultured under the condition of hypoxia or normal oxygen, which further showed that the transfection was successful.Invasion assay results showed that compared to untransfected group, after transfected si RNA-HIF-1α into 786-O cells, invasion of the cell number was respectively 43.7±1.2 v.s 48±1.6(3h);94±5.3 v.s 103±2.9(8h);148.7±12.4 v.s 146±12.9(12h); 278.7±23.1 vs 300±23(24h), P =0.84 > 0.05. si RNA-HIF-1α on cell invasion ability had no significant effect. But compared to untransfected group, after the si RNA-HIF-2αtransfected into renal cell carcinoma 786-O cells, the invasion of the cell number were43.7±1.2 v.s 40±0.8(3h);94±5.3 v.s 56±4.8(8h);148.7±12.4 v.s 100 ±16.3(12h); 278.7±23.1 v.s 183.3±47.1(24h). The results of single factor analysis of variance showed that P < 0.05, when the renal cell carcinoma 786-O cells were transfected with si RNA-HIF-2α, the invasion ability of the cells was no longer enhanced. It can be concluded that hypoxia induced invasioness of 786-O cells was enhanced by HIF 2α.3. Western blotting results showed that compared to the control group, in hypoxia group, the RCC 786-o cell protein expression(hypoxia v.s normoxia) are follows: HIF2α 0.73±0.066 v.s 0.157±0.046;COX2 0.90±0.096 v.s 0.085±0.028; E-cadherin 0.233±0.102 v.s 0.727±0.069;Snail 0.817±0.073 v.s 0.097±0.022. Compared to the control group, the experimental group(hypoxia) intracellular HIF2 α, COX2 expression increased. The expression of E-cadherin decreased and Snail expression increased(P < 0.05); HIF2α and COX2 expression levels were positively correlated(P = 0.001 < 0.05, r = 0.46), COX-2 and E-cadherin expression was negatively correlated with the level.(p=0.000 < 0.05, r=-0.34).Western blotting was used to detected the expression of proteins in renal cell carcinoma 786-O cell transfected with si RNA-HIF-1 α and si RNA-HIF-2α respectivly. The results showed that(si RNA-HIF-1α v.s si RNA-HIF-2α): COX2 0.93±0.20 v.s 0.07±0.03;E-cadherin 0.05±0.02 v.s 0.75±0.10;Snail 1.11±0.28 v.s 0.19±0.05. Single factor analysis of variance showed that P =0.000 < 0.05. Compared to the cells group without transfection the expression of intracellular HIF 2α was inhibited, which resulted in the intracellular expression of COX2 decreased and increased the expression of E-cadherin, Snail expression decreased, whereas inhibition of intracellular HIF-1α could not induce the changes of the protein expression.4. MTT assay showed that 786-O cells cultured for 12 hours under normal oxygen conditions, concentration of NS398 no more than 40 u M, cell proliferation activity(%) showed no significant change, 89±11.6 v.s 96±7.2,P>0.05. In order to exclude the direct toxic effect of NS398 on 786-O cells, the following experiments were used to select 40 u M concentration as the experimental concentration..5. Transwell assay results showed: in nomoxia group and nomoxia+NS398 group, cell invasion results were 114.3±26.6 v.s 117.7±26.3,P>0.05; hypoxia group and hypoxia +NS398: 154±17.7 v.s 110±26.9,P<0.05. Thus, under the condition of hypoxia, NS398 reversed the increased invasion ability of renal cell carcinoma 786-O.Western blot was used to detect the protein expression. HIF 2αexpression in normoxia group and normal oxygen group+NS398 group was follows: 0.16±0.06 v.s 0.11±0.03, P>0.05. The expression of HIF2α in hypoxia group and hypoxia+NS398 group: 0.73±0.06 v.s 0.73±0.03, P>0.05. COX2 expression in normoxia group and normoxia+NS398 group: 0.085±0.04 v.s 0.095±0.05, P>0.05, COX2 expression in hypoxia and hypoxia+NS398 group: 0.9±0.14 v.s 0.18±0.12,P<0.05. The expression of E-cadherin innormoxia and normoxia+NS398 group: 0.73±0.10 v.s 0.78±0.12,P>0.05. The expression of E-cadherin in hypoxia and hypoxia +NS398 group: 0.23±0.14 v.s 0.72±0.12,P<0.05. The expression of Snail in normoxia and normoxia+NS398 group: 0.10±0.03 v.s 0.09±0.05,P>0.05. The expression of Snail in hypoxia and hypoxia +NS398 group : 0.82±0.10 v.s 0.12±0.07,P<0.05. It can be seen that NS398 reduced the expression of COX2 in hypoxia 786-O cells, then the expression of E-cadherin was increased, and the expression of Snail decreased. Conclusion:1. Transient transfection was used to successfully transtected si-RNA-HIF 1 α and si-RNA-HIF 1 α into 786-O cells and the corresponding target protein could be suppressed effectively.2. Transfected with si-RNA-HIF 2 alpha, RCC 786-O cells cultured under hypoxic conditions, no longer appear cell invasion ability enhancement, and after transfected with si-RNA-HIF 1 α,786-O cells under hypoxic conditions, the invasion ability had no obvious change. It is concluded that the enhancement of the invasion ability of 786-O cells induced by hypoxia in renal cell carcinoma is regulated by HIF 2α.3. Although hypoxia induced HIF-1 α and HIF-2 α in renal cell carcinoma 786-O cells increases, but only HIF-2α can lead to increased the expression of target gene COX2 protein, eventually lead to E-cadherin/Snail pathway activation, resulting in enhanced cell invasioness. But increase of HIF-1α does not lead to activation of COX2 and E-cadehrin/Snail pathway.4. COX2 specific inhibitor NS398, can inhibit the expression of COX2 increase induced by hypoxia, resulting in inactivation of E-cadherin/Snail, so that hypoxia induced changes in the invasion ability of 786-O cells in the hypoxic environment no longer appear.Part Ⅲ Expression and significance of HIF2, COX2 and E-cadherin/Snail protein in renal cell carcinoma tissueObjective:To investigate the expression of HIF-2α, COX2 and E-cadherin/Snail in renal cell carcinoma, evaluated the relationship in the expression of these proteins and the relationship between the expression of these proteins and the clinical stage and/or pathological grading of renal cell carcinoma.Methods:From January 1, 2012---2015 years March 31, surgical resection of the 60 cases of renal cell carcinoma specimens were collected, according to 2004 WHO recomment and the 1997 who recommend about staging and clinincal staging, the pathological grading of these specimens were evaluated and the clinical staging of these patients were studied. Statistics of the patient’s age, gender, tumor size, clinical staging and pathological grading were recorded. immunohistochemical method was used to detect HIF2α, COX2 and E-cadherin/Snail protein expression in samples. These relationship in the proteins expression, between expression and grading or clinical staging was compared. Results:1. A total of 60 patients were diagnosed as renal cell carcinoma after operation. Of which 30 cases of renal clear cell carcinoma, 5 cases of chromophobe cell carcinoma, 4 cases of papillary carcinoma, collecting duct carcinoma in 1 cases. The oldest patient was 79 years old, the youngest was 26 years old, 29 males and 21 females. Ultrasonic examination of tumor diameter(the longest diameter) of the largest is 7.4cm, the smallest is 1.8cm. Clinical stage: stage I 15 cases, stage II 25 cases, stage III in 16 cases, 4 cases. In accordance with the pathological grade statistics of 14 cases were high differentiation, 27 cases of middle differentiation, 19 cases of low differentiation.2. All the specimens underwent immunohistochemical detection of HIF-2alpha, COX2 and E-cadherin and snail protein expression. HIF2 alpha positive expression in 47 cases(47/60) and negative expression in 11 patients(11/60). The positive expression of COX2 in 43 cases(43 / 60) and negative expression of 17 cases(17/60). The positive expression of E-cadherin in 25 cases(25 / 60) and negative expression in 35 cases(35 / 60); the positive expression of snail in 50 cases(50 / 60) and negative expression in 10 cases(10/60).3. Expression of HIF-2α is correlated with the expression of COX2 in renal cell carcinoma. With the increase of the expression intensity of HIF2α, the positive rate of COX2 expression in renal cell carcinoma was gradually increased, and the expression intensity was also increased.(P=0.001<0.05, r =0.442)4. There was a positive correlation between the expression of HIF-2α and the clinical stage of renal cell carcinoma. The positive rate of HIF-2α expression was higher in patients with higher clinical stage. In patients with higher clinical stage, the higher is the expression intensity of HIF2 alpha(P=0.000 < 0.05, r=0.439). The higher the grade of renal cell carcinoma was, the more likely it was to express HIF2 alpha, and the expression intensity was significantly correlated with the pathological grade(P=0.036<0.05,r=0.272).5. The expression of COX2 was inversely correlated with the expression of Ecadherin in renal cell carcinoma. The expression rate of E-cadherin was significantly lower in the positive expression of COX2. With the increase of COX2 expression intensity, the expression intensity of E-cadherin decreased gradually, and they were negatively correlated.6. With the clinical staging of patients getting higher, the expression rate and intensity of COX2 in renal cell carcinoma was increased. The expression of COX2 in renal cell carcinoma was positively correlated with the clinical stage(P=0.009 < 0.05, r=0.336) and pathological grading(P =0.018 < 0.05, r=0.303).7. In renal cell carcinoma, E-cadherin expression and the clinical stage had no significant correlation(P =0.073 > 0. 05); but in E-cadherin expression positive rate and expression intensity has negatively correlated with renal cell carcinoma pathological classification. And E-cadherin expression is lower, the tumor pathological grade may be higher(P = 0.006 < 0.05, r =-0.352). Conclusion:1. The expression of HIF2α in renal cell carcinoma was positively correlated with the expression of COX2, and the positive expression rate of COX2 was also increased with the increase of HIF2α positive expression rate.2. The expression of HIF-2α was positively correlated with the clinical stage and grade of the tumor.3. The expression of COX2 and E- cadherin(E-cadherin) was inversely correlated in renal cell carcinoma.4. The expression of COX2 was positively correlated with the clinical stage and pathological grade in renal cell carcinoma.5. In renal cell carcinoma, the expression of E-cadherin was not correlated with the clinical stage of the tumor, but was negatively correlated with the pathological grading.Part Ⅳ Effects of hypoxia and NS398 on Sorafenib IC50 in renal cell carcinoma 786-O cells Objective:To study the effect of hypoxia and NS398 on the IC50 of Sorafenib in 786-O cells. Methods:1. Renal cell carcinoma 786-O cells were divided into 8 groups, 4 groups were the experimental group, cultured under the condition of hypoxia. The control group was cultured under normal oxygen condition. Sorafenib and renal carcinoma cells were co-cultured for 12 hours according to 2.5u M, 5 u M, 10 u M, 20 u M final concentration. MTT colorimetric method was used to detect the proliferation of cells, and the inhibition rate of different concentrations of Sorafenib were calculated.2. The RCC 786-O cells were divided into hypoxia group, normoxia group, hypoxia group +NS398, normoxia+NS398 group. Each group again divided into four subgroups according to the different concentrations of sorafenib(2.5u M, 5.0 um, 10.0 um, 20.0 UM), incubation time is 12 h. MTT was used to detect the cell proliferation, and the inhibition rate of each group was calculated.According to the formula, cell inhibition rate(%) =1- cell activity(%), the cell inhibition rate was calculated. The Probit regression model was used in SPSS 19 software to analyze the IC50 cells of sorafenib in 786-O cells in the normoxia environment and the hypoxia environment.Results:1.MTT results:(hypoxic versus normoxic) 96.6±3.4 vs 78.9±4.41(2.5 u M);85.4±2.6 vs 62.4±2.4(5.0 u M);63±1.9 vs 42.9±2.5(10.0 u M);41.7±2.3 vs 17.2±1.6(20.0 u M). The cell proliferation in hypoxia group was significantly higher than that in the normal oxygen group, P < 0.05; IC50 Sorafenib were:(hypoxia v.s normal oxygen) 17.8±2.4u M v.s 7.8±1.5u M。P < 0.05。2. When renal cell carcinoma co-cultured with COX-2 specific inhibitor NS398 at concentration of 40 u M, the results of sorafenib were showed as: in the normoxic group IC50 was IC50 IC 50 7.25±2.17(u M), normoxia+NS398 group IC50 was 7.38±2.73(u M), hypoxia group IC50 15.01±3.19(u M), hypoxia +NS398 group IC50 was 6.99±1.98(u M). One factor analysis of variance showed that IC50 of hypoxia group was significantly higher than the other three groups(P < 0.05), but there was no significant difference between the normoxia group and normoxia+NS398 group and the hypoxia+NS398 group.Conclusion:1. The renal cell carcinoma 786-O cells cultured in hypoxic condition decreased the Sorafenib reaction, increase the drug tolerance the increase of IC50.2. Under anoxic conditions, Srafenib combined with NS398 cocultured with renal cell carcinoma 786-O cell, can effectively reduce the enhancement of Sorafenib IC50 induced by hypoxia.Part Ⅴ Effects of hypoxia on the growth of renal cell carcinoma in nude miceObjective:To investigate the effect of hypoxia on the growth of renal cell carcinoma in nude mice and the effect of hypoxia on the treatment of Sorafenib in nude mice of renal cancer model.Methods:1. Sixty male BALB / C nude mice of six week old were subcutaneously injected with 786-O cells, and then animals transplanted renal carcinoma model was established, the body weight and tumor size of nude mice were detected.2. When the tumor volume reached 40-50mm3, the animals were divided into 6 groups randomly(n=10). Three groups were fed under the condition of hypoxia,, the other 3 groups were fed under normal oxygen condition as control groups. This is to say, the hypoxia group, normoxia group, hypoxia+Sorafenib group, normoxia+Sorafenib group, hypoxia +NS398+Sorafenib group, normoxia+NS398+Sorafenib group were established. The dosage of Sorafenib was 30 mg/kg, 3mg/kg NS398 was used to gavage to nude mice, once a day. Control group was given the same volume of drug solvent. Measurement of tumor volume and body weight in nude mice every 2-3 days was executed.3. After 3 weeks of different, the mice were killed and the tumors were removed. The expression levels of HIF-2, COX2, E-cadherin, Snail and protein were detected by Western blotting.Results:1. Hypoxia and normoxia group, the tumor volume in nude mice began to appear obvious change after treatment of sorafenib at 9th day, tumor volume(hypoxic versus normoxic, mm3) were: 115.6±6.2 vs 109.5±8.1(9d);315.6±11.2 vs 228.5±7.2(12d);385.6±14.2 vs 346.6±12.6(15d);512.5±13.2 vs 459.6±12.3(18d);698.8±16.5 vs 626.3±16.2(21d), in various observation time, in hypoxia group, tumor volume of nude mice was significantly increased compared to normoxia group(P < 0.05).2. Normoxia group and normoxia+Sorafenib group, at each observation time point, tumor volume(hypoxic versus normoxic, mm3) respectively is: 109.5±8.1 vs 89.9±6.2(9d);228.5±7.2 vs 164.2±7.1(12d);346.6±12.6 vs 219.3±11.2(15d);459.0±15.2 vs 289.2±11.6(18d);626.3±16.2 vs 426.9±14.2(21d). At each observation time point, tumor volume of sorafenib treatment group(normoxia group +Sorafenib) was significantly reduced compared to the not drug treatment group.(P < 0.05).3. The tumor volum of hypoxia +Sorafenib, normoxia+Sorafenib at each observation time point were:(hypoxic versus normoxic, mm3) were: 108.4±5.8 vs 89.9±6.2(9d);208.6±5.2 vs 164.2±7.1(12d); 248.6±5.9 vs 219.3±11.2(15d);425.5±13.9 vs 289.2±11.6(18d);534.8±14.3 vs 426.9±14.2(21d). At each observation time point, the volume of tumor in nude mice were treated with targeted therapy under hypoxia were significantly increased(P < 0.05).4. In hypoxia+NS398+Sorafenib and normoxia+NS398+Sorafenib, tumor volume(hypoxic versus normoxic, mm3) were: 91.6±4.8 vs 81.9±6.2(9d);162.6±9.1 vs 135.5±7.2(12d); 178.2±3.1 vs 182.3±12.7(15d); 236.5±9.2 vs 245.0±12.4(18d);315.8±11.2 vs 326.8±12.9(21d). The tumor volume in the two groups had no significant change(P > 0.05).5. The expression of HIF2α, COX2, E-cadherin and Snail protein in tumor tissues was detected by Western blotting. The results showed that there was no significant difference in the expression of COX2, E-cadherin, snail and HIF2 in the hypoxia group. The expression of these protein was not significantly different between the normoxia groups. However, there was a significant difference in the expression of the protein between the hypoxia group and the normal oxygen group. In order to further study the cause of the change of tumor volume in the course of anti tumor therapy. Statistical analysis revealed that the expression of COX2 was correlated with the volume of tumor tissue, r=0.972. The results further showed that the COX2 specific inhibitor can effectively enhance the therapeutic efficacy of Sorafenib in the process of anti-tumor treament.Conclusions:1. Compared with normoxia condition, hypoxia can promote the growth of transplanted tumor in nude mice.2. The size of transplanted tumor in nude mice was positive related to the expression of COX2.3. Sorafenib can effectively inhibit the growth of tumor, but hypoxia can induce the tumor to decrease its reactivity to Sorafenib.4. NS398, a specific inhibitors of COX2 can enhance the anti-tumor effect of Sorafenib, reverse the tumor drug resistance induced by hypoxia. |
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