OBJECTIVE To develop a highly sensitive and specific quantitative real-time poly-merase chain reaction(qPCR)method for detecting the VGM-R02b(a gene therapy drug for glutaric acidemia type Ⅰ)gene in cynomolgus monkeys and analyze the biological distribution of VGM-R02b.METHODS A standard curve was constructed using the VGM-R02b standard plasmid[an adeno-associ-ated virus serotype 9(AAV9)capsid]on a qPCR platform.The detection method was optimized and validated for key parameters,including the quantitative range,accuracy,precision,dilution linearity,selectivity,specificity,stability,and parallelism.The established method was used to determine the target gene of VGM-R02b in the blood,brain,stomach,heart,liver,spleen,lung,kidney,thymus,and duodenum of cynomolgus monkeys on day 29(D29)and D92 after a single,unilateral intraventricular injection of VGM-R02b.The biodistribution of VGM-R02b in cynomolgus monkeys was analyzed.RESULTS A qPCR method for quantifying the VGM-R02b target gene in cynomolgus monkeys was established and validated.The standard curve demonstrated a quantitative range of 5.00×109 to 5.00×10^1 copies·μg-1 DNA,with excellent precision,accuracy,dilution linearity,selectivity,and specificity.The target gene in tissues remained stable after being stored at room temperature for 4 h,-15 to-25℃ for 9 d,-60 to-80℃ for 90 d and after five freeze-thaw cycles.Similarly,the target gene in whole blood remained stable after being stored at room temperature for 4 h,-15 to-25℃ for 93 d,-60 to-80℃ for 93 d and after five freeze-thaw cycles.Extracted nucleic acid samples also showed stability after being stored at room temperature for 4.25 h,2-8℃ for 24.16 h,-60 to-80℃ for 109 d and after five freeze-thaw cycles.The method was also applied to evaluate the biological distribution of the target gene in cynomolgus monkeys.In the control group,the target gene was undetectable on D29 and D92 post-administration,but in the drug administration group,the target gene was distributed across the tissues,with higher concen-trations observed in the brain,liver,spleen,and spinal cord,and there were no significant differences in the target gene content across the tissues between D29 and D92.CONCLUSION The established qPCR method is robust,reliable and suitable for determination of VGM-R02b target gene in cynomolgus monkeys.

OBJECTIVE To develop a highly sensitive and specific quantitative real-time poly-merase chain reaction(qPCR)method for detecting the VGM-R02b(a gene therapy drug for glutaric acidemia type Ⅰ)gene in cynomolgus monkeys and analyze the biological distribution of VGM-R02b.METHODS A standard curve was constructed using the VGM-R02b standard plasmid[an adeno-associ-ated virus serotype 9(AAV9)capsid]on a qPCR platform.The detection method was optimized and validated for key parameters,including the quantitative range,accuracy,precision,dilution linearity,selectivity,specificity,stability,and parallelism.The established method was used to determine the target gene of VGM-R02b in the blood,brain,stomach,heart,liver,spleen,lung,kidney,thymus,and duodenum of cynomolgus monkeys on day 29(D29)and D92 after a single,unilateral intraventricular injection of VGM-R02b.The biodistribution of VGM-R02b in cynomolgus monkeys was analyzed.RESULTS A qPCR method for quantifying the VGM-R02b target gene in cynomolgus monkeys was established and validated.The standard curve demonstrated a quantitative range of 5.00×109 to 5.00×10^1 copies·μg-1 DNA,with excellent precision,accuracy,dilution linearity,selectivity,and specificity.The target gene in tissues remained stable after being stored at room temperature for 4 h,-15 to-25℃ for 9 d,-60 to-80℃ for 90 d and after five freeze-thaw cycles.Similarly,the target gene in whole blood remained stable after being stored at room temperature for 4 h,-15 to-25℃ for 93 d,-60 to-80℃ for 93 d and after five freeze-thaw cycles.Extracted nucleic acid samples also showed stability after being stored at room temperature for 4.25 h,2-8℃ for 24.16 h,-60 to-80℃ for 109 d and after five freeze-thaw cycles.The method was also applied to evaluate the biological distribution of the target gene in cynomolgus monkeys.In the control group,the target gene was undetectable on D29 and D92 post-administration,but in the drug administration group,the target gene was distributed across the tissues,with higher concen-trations observed in the brain,liver,spleen,and spinal cord,and there were no significant differences in the target gene content across the tissues between D29 and D92.CONCLUSION The established qPCR method is robust,reliable and suitable for determination of VGM-R02b target gene in cynomolgus monkeys.

Objective To investigate the effect of the multimodal analgesia on postoperative pain after renal transplantation and the cytokines .Methods 40 cases of allogaft renal transplantation due to chronic renal failure were randomly divided into two groups (n=20) .The group D received the multimodal analgesia :preemptive analgesia plus patient controlled epidural analgesia(PCEA) and the group C(control) received analgesic drugs by intermittent intramuscular injection .The visual analogue scale(VAS) scores , the Ramsay sedation scores ,HR ,MAP and SPO2 at postoperative 2 ,6 ,12 ,24 ,48 h were recorded .Blood interleukin-2(IL-2) ,in-terleukin-6(IL-6) and interleukin-10(IL-10) levels were measured before anesthesia ,at the end of operation and postoperative 6 , 24 ,48 h .Results Postoperative MAP and SPO2 had no obvious change in the two groups ,no statistical differences in the various time points existed between the two groups (P>0 .05) .HR was significantly increased at 6 ,24 h after operation in the group C , which had statistical difference compared with that at the same time points in the group D (P<0 .05) .The VAS scores at postoper-ative 6 ,12 ,24 h in the group D were significantly lower than those in the group C ,the difference showed statistical significance (P<0 .05) .The sedation scores at various time points had no statistical difference between the two groups (P>0 .05) .The levels of IL-2 and IL-10 at postoperative 6 ,24 ,48 h in the two groups were significantly higher than those before anesthesia and at the end of operation (P<0 .05) .The levels of IL-2 and IL-6 at postoperative 6 ,24 ,48 h in the group D were significantly lower than those in the group C(P<0 .05) .Conclusion Multimodal analgesia can reach the effective analgesic effect ,down-regulate the pro-inflam-matory cytokines and up-regulate anti-inflammatory cytokines for maintaining postaperative serum cytokines balance .

BACKGROUND:Multimodal analgesia provides sufficient analgesia in renal recipients and appears to be associated with the recovery of renal function after transplantation. OBJECTIVE:To investigate the effect of multimodal analgesia with dezocine on postoperative immunity after renal transplantation, and discuss the appropriate analgesic drugs and methods for patients with renal transplantation. METHODS:Forty patients undergoing renal transplantation were randomly divided into two groups. They al received general anesthesia combined with epidural blockage. Control group received intramuscular injection of analgesic drugs when needed, while dezocine group received multimodal analgesia:preemptive anaIgesia with dezocine+patient-control ed epidural analgesia. The heart rate, mean arterial pressure, and saturation of blood oxygen were detected before anesthesia, 12, 24, 48 hours after transplantation. T lymphocyte subsets, interleukin-2, interleukin-6 and interleukin-10 levels in venous blood were measured before anesthesia, 12, 24, 48 hours after transplantation. RESULTS AND CONCLUSION:Compared with before anesthesia, the CD4+, CD8+cellsubset counts, CD4+/CD8+ratio, the levels of interleukin-2 and interleukin-6 were decreased significantly (P<0.05), and the levels of interleukin were significantly increased after transplantation in the control group (P<0.05). The postoperative CD4+cellsubset counts, the levels of interleukin-2 and interleukin-6 were significantly lower at 12 hours after transplantation than that before anesthesia (P<0.05), then recovered to normal levels at 24 hours in dezocine group. The postoperative CD8+cellsubset counts, CD8+and CD4+/CD8+ratio were not changed before and after transplantation in the dezocine group. The levels of interleukin-10 in the dezocine group were significantly increased at 48 hours after transplantation compared with before anesthesia (P<0.05), which was stil lower than that in control group (P<0.05). Multimodal analgesia with dezocine can effectively protect the immune system, promote short-term turnover of renal function, and prolong graft survival for patients with renal transplantation.

BACKGROUND: Serious lesion to liver function may cause the pulmonary artery hypertension and fluctuation of haemodynamics directly induces changes of pulmonary artery pressure during neo-hepatic stage in liver transplantation. OBJECTIVE: To observe the change of pulmonary artery pressure during liver transplantation and to explore treatment for pulmonary artery hypertension. DESIGN, TIME AND SETTING: Controlled observation was carried out among the liver transplant recipients who were enrolled from the 181 Hospital of Chinese PLA from June 2004 to June 2006. PARTICIPANTS: Totally 13 patients, all males, were reviewed. Their ages ranged from 19 to 53 years. They were selective for the liver transplantation. METHODS: Undergoing general anesthesia, mean arterial blood pressure, central venous pressure, pulmonary arterial pressure and pulmonary arterial wedge pressure were continuously monitored by Swan-Ganz catheter method and reversible Fick’s law continuous monitoring. Available respiration administration, low dose of glyceryltrinitrate (0.1-5.0 ?g/kg/min) and Alprostadil (1.0-2.0 ?g) were administered to diminish elevated pulmonary arterial pressure and pulmonary arterial wedge pressure during the period after unclamping portal vein in liver transplantation. MAIN OUTCOME MEASURES: Changes of mean arterial blood pressure and pulmonary arterial pressure. RESULTS: The mean arterial blood pressure deceased at the time of unclamping portal vein in all 13 patients, pulmonary arterial pressure and pulmonary arterial wedge pressure both increased significantly at 5 and 15 minutes within neo-hepatic stage, which were more obvious than those during anhepatic stage in 13 patients (P

BACKGROUND: Serious lesion to liver function may cause the pulmonary artery hypertension and fluctuation of haemodynamics directly induces changes of pulmonary artery pressure during neo-hepatic stage in liver transplantation. OBJECTIVE: To observe the change of pulmonary artery pressure during liver transplantation and to explore treatment for pulmonary artery hypertension. DESIGN, TIME AND SETTNG: Controlled observation was carried out among the liver transplant recipients who were enrolled from the 181 Hospital of Chinese PLA from June 2004 to June 2006.PARTICIPANTS: Totally 13 patients, all males, were reviewed. Their ages ranged from 19 to 53 years. They were selective for the liver transplantation. METHODS: Undergoing general anesthesia, mean arterial blood pressure, central venous pressure, pulmonary arterial pressure and pulmonary arterial wedge pressure were continuously monitored by Swan-Ganz catheter method and reversible Fick's law continuous monitoring. Available respiration administration, low dose of glyceryltrinitrate (0.1-5.0 u g/kg/min) and Alprostadil (1.0-2.0 u g) were administered to diminish elevated pulmonary arterial pressure and pulmonary arterial wedge pressure during the period after unclamping portal vein in liver transplantation. MAIN OUTCOME MEASURES: Changes of mean arterial blood pressure and pulmonary arterial pressure. RESULTS: The mean arterial blood pressure deceased at the time of unclamping portal vein in all 13 patients, pulmonary arterial pressure and pulmonary arterial wedge pressure both increased significantly at 5 and 15 minutes within neo-hepatic stage, which were more obvious than those during anhepatic stage in 13 patients(P＜0.05).The pulmonary arterial pressure and pulmonary arterial wedge pressure revived to the level before unclamping inferior vein by treatment with glyceryltrinitrate and Alprostadil within 30 minutes. CONCLUSION: Reasonable application of glyceryltrinitrate and Alprostadil can obviously release pulmonary arterial pressure during neo-hepatic stage in liver transplantation.