To assess the efficacy and the optimum dose of recombinant human erythropoietin (rhEpo) on the anemia of premature, 45 preterm infants with a gestational age of less than 35 weeks and birth weight of less 1 800 g were...To assess the efficacy and the optimum dose of recombinant human erythropoietin (rhEpo) on the anemia of premature, 45 preterm infants with a gestational age of less than 35 weeks and birth weight of less 1 800 g were randomly assigned to treatment group 1 (n = 15, receiving subcutaneous rhEpo 150 U/kg·time), treatment group 2 (n = 15, receiving 250 U/kg·time), three times a week for 6 weeks, and control group (n = 15, no treatment was given). All preterm infants received supplements of vitamin E (20 IU) and iron (20 mg) each day. Our results showed that postnatal decline of hemoglobin (Hb) and hematocrit (Hct) were lessened in the treatment groups, particularly in the group 2 and the differences were very significant (P<0. 0001 for all). Treated infants had significantly higher reticulocyte counts (Ret) (P<0. 000] for all), but there was no significant difference between the two treatment groups (P>0. 05). Serum iron dropped significantly in the treatment groups as compared with control group (P<0. 01 for all), but no dose-dependent relationship was observed in treated infants (P>0. 05). After treatment, serum levels of erythropoietin was higher in group 2 than those in group 1 and control group (P<0. 0001, P<0. 01 and P<0. 05, respectively). There was no significant difference between group 1 and control group (P>0. 05). No side effects related to rhEpo therapy were observed. Our study suggested that rhEpo therapy stimulates endogenous erythro-poiesis and enhances Ret, Hct and level of Hb in a dose-dependent manner in premature infants. The therapy is more efficient when given in higher dosages.展开更多
BACKGROUND Hema-Plus,a recombinant human erythropoietin(rHuEPO)or epoetin alfa has shown effectiveness in correction of anemia in Thai population in clinical practice.This study was aimed to demonstrate efficacy and s...BACKGROUND Hema-Plus,a recombinant human erythropoietin(rHuEPO)or epoetin alfa has shown effectiveness in correction of anemia in Thai population in clinical practice.This study was aimed to demonstrate efficacy and safety under the evidencebased approach.AIM To evaluate the efficacy and safety of rHuEPO(Hema-Plus)for treatment of anemia over 12 wk in Thai patients with Stage V chronic kidney disease(CKD)on peritoneal dialysis(PD).METHODS This study was an open-label,multi-center study to enroll 30 CKD patients identified to start PD with hemoglobin(Hb)less than 9.5 g/dL,serum ferritin more than 100 ng/mL,serum transferrin saturation more than or equal to 20%and who had not previously received epoetin.Patients with conditions that could increase the risk of adverse effects from study participation or interfere with study outcomes,were using concomitant androgens or had secondary hyperparathyroidism were excluded.All eligible patients started Hema-Plus by SC injection at 4000 IU once or twice weekly(week 0)and with follow-up at weeks 2,4,8,and 12.Dosage adjustment could be done to achieve Hb level of 11-12 g/dL.Primary end point was mean change in Hb level from baseline to end of treatment(week 12).Safety was assessed throughout the study.Quality of life(QoL)was assessed using KDQOL-36.RESULTS All 30 enrolled patients completed the study.Mean(standard deviation)Hb at baseline(week 0)to the end of 12 wk was significantly increased from 7.39(1.29)g/dL to 11.15(1.73)g/dL(paired t-test,P value<0.001).Overall change of Hb means from baseline over the other 4 visits was statistically significantly increased(repeated measure ANOVA,P value<0.001).Ten out of 39 adverse events(AEs)were serious.Two serious AEs were probably related to study medication by investigators’assessment.At week 12,the QoL scores in all domains were significantly increased from baseline.CONCLUSION Hema-Plus administered for 12 wk for treatment of anemia in patients on PD effectively increased Hb levels with acceptable safety profile.展开更多
Objective To produce specific monoclonal antibody （mAb） against recombinant human erythropoietin （rHuEPO） for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuE...Objective To produce specific monoclonal antibody （mAb） against recombinant human erythropoietin （rHuEPO） for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuEPO was covalently coupled with bovine serum albumin （BSA） and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked irmnunosorbent assay （ELISA）, SDS-PAGE and Western blot. Results The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1x10s L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.展开更多
Objective To observe the expression of hypoxia inducible factor-1α (HIF-1α) in the retina of rabbits with acute high intraocular pressure and to investigate the mechanism of systemic domestic recombinant human eryth...Objective To observe the expression of hypoxia inducible factor-1α (HIF-1α) in the retina of rabbits with acute high intraocular pressure and to investigate the mechanism of systemic domestic recombinant human erythropoietin (rhEPO) protecting the retina from ischemia-reperfusion injury. Methods First,control group and model group were established in rabbit eyes. The acute high intraocular pressure model was established by saline perfusion into anterior chamber,and then hypodermic injection of domestic rhEPO was made. HIF-1α protein in the retina was observed by immunohistochemical staining method on days 1,3,7 and 14 after retinal ischemia-reperfusion,respectively. Results No cells with HIF-1α positive expression were observed in the retina of the control group. Cells with HIF-1α positive expression in the model group outnumbered those in the control group (P<0.01). The resemblance pattern occurred in EPO group but its degree was slightly greater than that in the model group from day 3 after ischemia-reperfusion (P<0.05). Conclusion Domestic rhEPO can down-regulate the expression of HIF-1α in the retina with acute high intraocular pressure,which may be one of the mechanisms that rhEPO protects the retina from ischemia-reperfusion injury.展开更多
Two different immunoassay methods, competitive indirect enzyme-linked immuno-sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect ...Two different immunoassay methods, competitive indirect enzyme-linked immuno-sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO. The linear ranges were 50-20000 ng/mL and 10-50000 ng/mL for CI-ELISA and ACI-ELISA, respectively. The low detection limits of CI-ELISA and ACI-ELISA were 62.8 ng/mL and 8.5 ng/mL, respectively.展开更多
AIM:Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron,but patients usually face a two-month recovery period from post- haemorrhage anaemia.This prospective,randomised,op...AIM:Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron,but patients usually face a two-month recovery period from post- haemorrhage anaemia.This prospective,randomised,open, pilot study was designed to investigate whether recombinant human erythropoietin(Epoetin)therapy accelerate haematocrit increase in the post-bleeding recovery period. METHODS:We studied hospitalised patients admitted because of acute ulcer bleeding or haemorrhagic gastritis, who had a haematocrit of 27-33% and did not receive blood transfusions.One day after the endoscopic confirmation of cessation of bleeding,they were randomised either to erythropoietin(20 000 IU Epoetin alfa subcutaneously,on days 0,4 and 6)plus iron(100 mg im,on days 1-6,(G_1)or iron only(G_2).Haematocdt was measured on days 0,6,14, 30,45,and 60,respectively. RESULTS:One patient from G_1 and two from G_2 were lost to follow-up.Therefore,14 and 13 patients from G_1 and G_2 respectively were analysed.Demographic characteristics,serum iron,ferritin,total iron binding capacity,reticulocytes,and haernatoait were not significantly different at entry to the study. Median reticulocyte counts were significantly different between groups on day six(G_1:4.0,3.0-6.4 vs G_2:3.5,2.1-4.4%, P=0.03)and median haematocrit on day fourteen [G_1:35.9, 30.7-41.0 vs G_2:32.5,29.5-37.0%(median,range),P=0.04]. CONCLUSION:Erythropoietin administration significantly accelerates correction of anemia after acute ulcer bleeding. The haematocrit gain is equivalent to one unit of transfused blood two weeks after the bleeding episode.展开更多
Objective: To explore the regulative effect on Survivin of r-HuEPO after traumatic brain injury (TBI) in rats, and understand the neuroprotection mechanisms of r-HuEPO. Methods: Seventy-eight adult Wistar rats were ra...Objective: To explore the regulative effect on Survivin of r-HuEPO after traumatic brain injury (TBI) in rats, and understand the neuroprotection mechanisms of r-HuEPO. Methods: Seventy-eight adult Wistar rats were randomly divided into sham operation group (n = 6), TBI group (n = 36) and r-HuEPO group (n = 36). The experimental TBI model was created by Feeney’s method. Samples were obtained after injury for measuring apoptosis of cells by Epics XL Flow Cytometer. Immunochemical method was performed for inspection of expressions of Survivin and NF-κB proteins. Results: Compared to the sham group, the number of apoptotic cells and Survivin, NF-κB immunopositive cells was significantly increased in the injured brain after TBI (P < 0.01). R-HuEPO significantly increased the expression of survivin and NF-κB, but decreased the apoptotic rates. Conclusion: Increased expression of NF-κB by r-HuEPO may play important role in regulating Survivin level, inhibiting neuronal apoptosis in cortex and exerting protective function to neurons.展开更多
目的:探讨重组人促红细胞生成素(rHuEPO)对大鼠皮肤切创形成的急性创面愈合过程中血管生成素-1(Ang-1)表达的影响。方法:健康SD雄性72只大鼠建立急性创面动物模型,随机分成低剂量组(50 U/m L rHu EPO),中剂量组(100 U/m L rHuEPO),高剂...目的:探讨重组人促红细胞生成素(rHuEPO)对大鼠皮肤切创形成的急性创面愈合过程中血管生成素-1(Ang-1)表达的影响。方法:健康SD雄性72只大鼠建立急性创面动物模型,随机分成低剂量组(50 U/m L rHu EPO),中剂量组(100 U/m L rHuEPO),高剂量组(150 U/m L rHuEPO)和生理盐水(NS)对照组,造模同时进行外敷不同剂量rHuEPO及生理盐水治疗。观察每组大鼠创面愈合情况,并于伤后3、7、14 d取创面皮肤标本,采用免疫组织化学方法染色检测创面CD_(31)、Ang-1的表达。结果:免疫组化结果为CD_(31)、Ang-1在高剂量和中剂量药物治疗组皮肤组织中高表达,在同一时相点与NS对照组、低剂量组有统计学差异(P<0.05)。结论:rHuEPO能促进大鼠皮肤急性创面愈合过程中Ang-1表达,并促进创面微血管形成,从而加速急性创面愈合。展开更多
目的临床治疗虽可延缓肾间质纤维化进展,却无法逆转肾功能丧失。探讨重组人促红细胞生成素(recombinant human erythropoietin,r Hu EPO)对肾间质纤维化过程中炎症因子的影响及其可能作用机制。方法将体外培养的HK-2细胞随机分为7组:空...目的临床治疗虽可延缓肾间质纤维化进展,却无法逆转肾功能丧失。探讨重组人促红细胞生成素(recombinant human erythropoietin,r Hu EPO)对肾间质纤维化过程中炎症因子的影响及其可能作用机制。方法将体外培养的HK-2细胞随机分为7组:空白对照组、r Hu EPO对照组(20 U/m L r Hu EPO)、清蛋白刺激组(5 mg/m L清蛋白)、5 U/m L r Hu EPO干预组(5 mg/m L清蛋白+5 U/m L r Hu EPO)、10 U/m L r Hu EPO干预组(5 mg/m L清蛋白+10 U/m L r Hu EPO)、20 U/m L r Hu EPO干预组(5 mg/m L清蛋白+20 U/m L r Hu EPO)、Rho激酶抑制组(10μmol/L Y27632+5 mg/m L清蛋白),各组均作用24 h。观察各组细胞形态的变化;RT-PCR检测各组细胞Rho A、ROCK1 mRNA及白细胞介素-6因子(interleukin-6,IL-6)mRNA含量水平;ELISA检测细胞上清液中肿瘤坏死因子(tumor necrosis factor,TNF-α)、IL-6蛋白的含量表达。结果空白对照组、r Hu EPO干预组显示鹅卵石或者铺路石样形态,清蛋白刺激组表现出细长梭状改变,呈现纤维细胞样外观。5、10、20 U/m L r Hu EPO干预组细胞向鹅卵石样复转,Rho激酶抑制组细胞形态呈椭圆形、细胞间隙稍增大;与空白对照组比较,清蛋白刺激组Rho A、ROCK1 mRNA及IL-6 mRNA显著升高(P<0.05),而5、10、20 U/m L r Hu EPO干预组逐渐下调(P<0.05),且与r Hu EPO浓度负相关;与清蛋白刺激组比较,Rho激酶抑制组ROCK1 mRNA、IL-6 mRNA表达下调(P<0.05),但Rho A mRNA表达差异无统计学意义(P>0.05)。ELISA结果显示:清蛋白刺激组上清液TNF-α、IL-6蛋白[(1 347.54±41.52)ng/L、(884.62±0.73)pg/L]表达较空白对照组[(452.32±33.23)ng/L,(95.12±0.32)pg/L]显著增高(P<0.05),5、10、20 U/m L r Hu EPO干预组、Rho激酶抑制组TNF-α表达[(1 003.32±3.42)、(821.32±21.32)、(590.15±7.68)、(488.13±65.03)ng/L)]较清蛋白刺激组[(1 347.54±41.52)ng/L]下降(P<0.05)、IL-6蛋白表达[(656.68±0.55)、(422.35±0.22)、(217.32±0.35)、(309.49±0.21)pg/L]亦较清蛋白刺激组[(884.62±0.73)pg/L]下降(P<0.05),5、10、20 U/m L r Hu EPO干预组组间两两比较差异均有统计学意义(P<0.05)。结论 r Hu EPO可能通过减少炎症因子的产生来抑制清蛋白诱导的HK-2细胞转分化过程,其作用机制部分涉及Rho A/ROCK信号通路。展开更多
文摘To assess the efficacy and the optimum dose of recombinant human erythropoietin (rhEpo) on the anemia of premature, 45 preterm infants with a gestational age of less than 35 weeks and birth weight of less 1 800 g were randomly assigned to treatment group 1 (n = 15, receiving subcutaneous rhEpo 150 U/kg·time), treatment group 2 (n = 15, receiving 250 U/kg·time), three times a week for 6 weeks, and control group (n = 15, no treatment was given). All preterm infants received supplements of vitamin E (20 IU) and iron (20 mg) each day. Our results showed that postnatal decline of hemoglobin (Hb) and hematocrit (Hct) were lessened in the treatment groups, particularly in the group 2 and the differences were very significant (P<0. 0001 for all). Treated infants had significantly higher reticulocyte counts (Ret) (P<0. 000] for all), but there was no significant difference between the two treatment groups (P>0. 05). Serum iron dropped significantly in the treatment groups as compared with control group (P<0. 01 for all), but no dose-dependent relationship was observed in treated infants (P>0. 05). After treatment, serum levels of erythropoietin was higher in group 2 than those in group 1 and control group (P<0. 0001, P<0. 01 and P<0. 05, respectively). There was no significant difference between group 1 and control group (P>0. 05). No side effects related to rhEpo therapy were observed. Our study suggested that rhEpo therapy stimulates endogenous erythro-poiesis and enhances Ret, Hct and level of Hb in a dose-dependent manner in premature infants. The therapy is more efficient when given in higher dosages.
基金the Ethics Committee,Institute for the Development of Human Research Protections(IHRP),No.IHRP 2365/2556The Ethics Committee of Udon Thani Hospital,No.8/2559and Siriraj Institutional Review Board Faculty of Medicine Siriraj Hospital,Mahidol University,No.Si 588/2016.
文摘BACKGROUND Hema-Plus,a recombinant human erythropoietin(rHuEPO)or epoetin alfa has shown effectiveness in correction of anemia in Thai population in clinical practice.This study was aimed to demonstrate efficacy and safety under the evidencebased approach.AIM To evaluate the efficacy and safety of rHuEPO(Hema-Plus)for treatment of anemia over 12 wk in Thai patients with Stage V chronic kidney disease(CKD)on peritoneal dialysis(PD).METHODS This study was an open-label,multi-center study to enroll 30 CKD patients identified to start PD with hemoglobin(Hb)less than 9.5 g/dL,serum ferritin more than 100 ng/mL,serum transferrin saturation more than or equal to 20%and who had not previously received epoetin.Patients with conditions that could increase the risk of adverse effects from study participation or interfere with study outcomes,were using concomitant androgens or had secondary hyperparathyroidism were excluded.All eligible patients started Hema-Plus by SC injection at 4000 IU once or twice weekly(week 0)and with follow-up at weeks 2,4,8,and 12.Dosage adjustment could be done to achieve Hb level of 11-12 g/dL.Primary end point was mean change in Hb level from baseline to end of treatment(week 12).Safety was assessed throughout the study.Quality of life(QoL)was assessed using KDQOL-36.RESULTS All 30 enrolled patients completed the study.Mean(standard deviation)Hb at baseline(week 0)to the end of 12 wk was significantly increased from 7.39(1.29)g/dL to 11.15(1.73)g/dL(paired t-test,P value<0.001).Overall change of Hb means from baseline over the other 4 visits was statistically significantly increased(repeated measure ANOVA,P value<0.001).Ten out of 39 adverse events(AEs)were serious.Two serious AEs were probably related to study medication by investigators’assessment.At week 12,the QoL scores in all domains were significantly increased from baseline.CONCLUSION Hema-Plus administered for 12 wk for treatment of anemia in patients on PD effectively increased Hb levels with acceptable safety profile.
基金This work was supported by the National Natural Science Foundation of China (No.20675006).
文摘Objective To produce specific monoclonal antibody （mAb） against recombinant human erythropoietin （rHuEPO） for development of highly efficient methods for erythropoietin detection in biological fluids. Methods rHuEPO was covalently coupled with bovine serum albumin （BSA） and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked irmnunosorbent assay （ELISA）, SDS-PAGE and Western blot. Results The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1x10s L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work. Conclusions The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.
基金supported by the Science and Technology Research Foundation of Shaanxi Province, China (No.2008K14-07)the Medical and Phar macologic Research Foundation of Xi'an City, China (No.SF08004-2)
文摘Objective To observe the expression of hypoxia inducible factor-1α (HIF-1α) in the retina of rabbits with acute high intraocular pressure and to investigate the mechanism of systemic domestic recombinant human erythropoietin (rhEPO) protecting the retina from ischemia-reperfusion injury. Methods First,control group and model group were established in rabbit eyes. The acute high intraocular pressure model was established by saline perfusion into anterior chamber,and then hypodermic injection of domestic rhEPO was made. HIF-1α protein in the retina was observed by immunohistochemical staining method on days 1,3,7 and 14 after retinal ischemia-reperfusion,respectively. Results No cells with HIF-1α positive expression were observed in the retina of the control group. Cells with HIF-1α positive expression in the model group outnumbered those in the control group (P<0.01). The resemblance pattern occurred in EPO group but its degree was slightly greater than that in the model group from day 3 after ischemia-reperfusion (P<0.05). Conclusion Domestic rhEPO can down-regulate the expression of HIF-1α in the retina with acute high intraocular pressure,which may be one of the mechanisms that rhEPO protects the retina from ischemia-reperfusion injury.
文摘Two different immunoassay methods, competitive indirect enzyme-linked immuno-sorbent assay (CI-ELISA) and amplificative competitive indirect ELISA (ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO. The linear ranges were 50-20000 ng/mL and 10-50000 ng/mL for CI-ELISA and ACI-ELISA, respectively. The low detection limits of CI-ELISA and ACI-ELISA were 62.8 ng/mL and 8.5 ng/mL, respectively.
文摘AIM:Anaemia caused by acute upper gastrointestinal bleeding is treated with blood transfusion or iron,but patients usually face a two-month recovery period from post- haemorrhage anaemia.This prospective,randomised,open, pilot study was designed to investigate whether recombinant human erythropoietin(Epoetin)therapy accelerate haematocrit increase in the post-bleeding recovery period. METHODS:We studied hospitalised patients admitted because of acute ulcer bleeding or haemorrhagic gastritis, who had a haematocrit of 27-33% and did not receive blood transfusions.One day after the endoscopic confirmation of cessation of bleeding,they were randomised either to erythropoietin(20 000 IU Epoetin alfa subcutaneously,on days 0,4 and 6)plus iron(100 mg im,on days 1-6,(G_1)or iron only(G_2).Haematocdt was measured on days 0,6,14, 30,45,and 60,respectively. RESULTS:One patient from G_1 and two from G_2 were lost to follow-up.Therefore,14 and 13 patients from G_1 and G_2 respectively were analysed.Demographic characteristics,serum iron,ferritin,total iron binding capacity,reticulocytes,and haernatoait were not significantly different at entry to the study. Median reticulocyte counts were significantly different between groups on day six(G_1:4.0,3.0-6.4 vs G_2:3.5,2.1-4.4%, P=0.03)and median haematocrit on day fourteen [G_1:35.9, 30.7-41.0 vs G_2:32.5,29.5-37.0%(median,range),P=0.04]. CONCLUSION:Erythropoietin administration significantly accelerates correction of anemia after acute ulcer bleeding. The haematocrit gain is equivalent to one unit of transfused blood two weeks after the bleeding episode.
文摘Objective: To explore the regulative effect on Survivin of r-HuEPO after traumatic brain injury (TBI) in rats, and understand the neuroprotection mechanisms of r-HuEPO. Methods: Seventy-eight adult Wistar rats were randomly divided into sham operation group (n = 6), TBI group (n = 36) and r-HuEPO group (n = 36). The experimental TBI model was created by Feeney’s method. Samples were obtained after injury for measuring apoptosis of cells by Epics XL Flow Cytometer. Immunochemical method was performed for inspection of expressions of Survivin and NF-κB proteins. Results: Compared to the sham group, the number of apoptotic cells and Survivin, NF-κB immunopositive cells was significantly increased in the injured brain after TBI (P < 0.01). R-HuEPO significantly increased the expression of survivin and NF-κB, but decreased the apoptotic rates. Conclusion: Increased expression of NF-κB by r-HuEPO may play important role in regulating Survivin level, inhibiting neuronal apoptosis in cortex and exerting protective function to neurons.
文摘目的:探讨重组人促红细胞生成素(rHuEPO)对大鼠皮肤切创形成的急性创面愈合过程中血管生成素-1(Ang-1)表达的影响。方法:健康SD雄性72只大鼠建立急性创面动物模型,随机分成低剂量组(50 U/m L rHu EPO),中剂量组(100 U/m L rHuEPO),高剂量组(150 U/m L rHuEPO)和生理盐水(NS)对照组,造模同时进行外敷不同剂量rHuEPO及生理盐水治疗。观察每组大鼠创面愈合情况,并于伤后3、7、14 d取创面皮肤标本,采用免疫组织化学方法染色检测创面CD_(31)、Ang-1的表达。结果:免疫组化结果为CD_(31)、Ang-1在高剂量和中剂量药物治疗组皮肤组织中高表达,在同一时相点与NS对照组、低剂量组有统计学差异(P<0.05)。结论:rHuEPO能促进大鼠皮肤急性创面愈合过程中Ang-1表达,并促进创面微血管形成,从而加速急性创面愈合。
文摘目的临床治疗虽可延缓肾间质纤维化进展,却无法逆转肾功能丧失。探讨重组人促红细胞生成素(recombinant human erythropoietin,r Hu EPO)对肾间质纤维化过程中炎症因子的影响及其可能作用机制。方法将体外培养的HK-2细胞随机分为7组:空白对照组、r Hu EPO对照组(20 U/m L r Hu EPO)、清蛋白刺激组(5 mg/m L清蛋白)、5 U/m L r Hu EPO干预组(5 mg/m L清蛋白+5 U/m L r Hu EPO)、10 U/m L r Hu EPO干预组(5 mg/m L清蛋白+10 U/m L r Hu EPO)、20 U/m L r Hu EPO干预组(5 mg/m L清蛋白+20 U/m L r Hu EPO)、Rho激酶抑制组(10μmol/L Y27632+5 mg/m L清蛋白),各组均作用24 h。观察各组细胞形态的变化;RT-PCR检测各组细胞Rho A、ROCK1 mRNA及白细胞介素-6因子(interleukin-6,IL-6)mRNA含量水平;ELISA检测细胞上清液中肿瘤坏死因子(tumor necrosis factor,TNF-α)、IL-6蛋白的含量表达。结果空白对照组、r Hu EPO干预组显示鹅卵石或者铺路石样形态,清蛋白刺激组表现出细长梭状改变,呈现纤维细胞样外观。5、10、20 U/m L r Hu EPO干预组细胞向鹅卵石样复转,Rho激酶抑制组细胞形态呈椭圆形、细胞间隙稍增大;与空白对照组比较,清蛋白刺激组Rho A、ROCK1 mRNA及IL-6 mRNA显著升高(P<0.05),而5、10、20 U/m L r Hu EPO干预组逐渐下调(P<0.05),且与r Hu EPO浓度负相关;与清蛋白刺激组比较,Rho激酶抑制组ROCK1 mRNA、IL-6 mRNA表达下调(P<0.05),但Rho A mRNA表达差异无统计学意义(P>0.05)。ELISA结果显示:清蛋白刺激组上清液TNF-α、IL-6蛋白[(1 347.54±41.52)ng/L、(884.62±0.73)pg/L]表达较空白对照组[(452.32±33.23)ng/L,(95.12±0.32)pg/L]显著增高(P<0.05),5、10、20 U/m L r Hu EPO干预组、Rho激酶抑制组TNF-α表达[(1 003.32±3.42)、(821.32±21.32)、(590.15±7.68)、(488.13±65.03)ng/L)]较清蛋白刺激组[(1 347.54±41.52)ng/L]下降(P<0.05)、IL-6蛋白表达[(656.68±0.55)、(422.35±0.22)、(217.32±0.35)、(309.49±0.21)pg/L]亦较清蛋白刺激组[(884.62±0.73)pg/L]下降(P<0.05),5、10、20 U/m L r Hu EPO干预组组间两两比较差异均有统计学意义(P<0.05)。结论 r Hu EPO可能通过减少炎症因子的产生来抑制清蛋白诱导的HK-2细胞转分化过程,其作用机制部分涉及Rho A/ROCK信号通路。
