OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique. METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method. RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice. CONCLUSIONS We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.
Animals ; CRISPR-Cas Systems ; Female ; Genetic Vectors ; Hepatitis B e Antigens ; genetics ; Hepatitis B virus ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic

Objective To observe the effect of continuous pumped dexmedetomidine on hemodynamics, awakening quality and stress response in double -lumen tube extubation patients.Methods 60 patients with thoracic surgery were divided into saline group(group N,n =30)and Dex group(group D,n =30).Both two groups were continuously pumped NS and dexmedetomidine respectively 20min before anesthesia.MAP,HR and SpO2 at different time points during the surgery were recorded.Extracted 5ml arterial blood at the time point of T0 and T3,centrifugated and stored at -80 ℃ for testing renin levels.The extubation time,restlessness incidence,orientation recovery time, extubation quality score were recorded.Results The values of MAP in group D at time points of T0,T1,T2,T3,T4 were (97 ±15)mmHg,(102 ±12)mmHg,(103 ±11)mmHg,(98 ±12)mmHg,(96 ±13)mmHg.The values of HR in group D at different time points were (69 ±9)times/min,(80 ±6)times/min,(90 ±7)times/min,(74 ±5)times/min,(73 ±6)times/min.The values of SpO2 in group D at different time points were (95 ±3)%,(98 ±2)%,(98 ± 2)%,(95 ±3)%,(96 ±2)%.The values of MAP in group N at time points of T0,T1,T2,T3,T4 were (96 ± 15)mmHg,(112 ±16)mmHg,(120 ±12)mmHg,(117 ±14)mmHg,(102 ±15)mmHg.The values of HR in group N at different time points were (68 ±7)times/min,(84 ±8)times/min,(105 ±6)times/min,(89 ±6)times/min, (80 ±7)times/min.The values of SpO2 in group N at different time points were (96 ±2)%,(98 ±2)%,(97 ± 3)%,(93 ±3)%,(92 ±4)%.The values of MAP,HR,SpO2 at different time points in group D were significantly lower than those in group N,the differences were statistically significant (the t values of MAP at different time points were tT1 =2.74,tT2 =5.72,tT3 =25.63,tT4 =1.66,all P <0.05;the t values of HR at different time points were tT1 =2.20,tT2 =8.91,tT3 =10.52,tT4 =4.16,all P <0.05;the t values of SpO2 at different time points were tT1 =0.00,tT2 =1.52,tT3 =2.58,tT4 =4.9,PT3 <0.05,PT4 <0.05).There was significant difference between the two groups in orientation recovery time[(3.3 ±2.2)min vs (5.2 ±2.3)min,P =0.0018].There was significant difference between the two groups in agitation score[(1.3 ±0.9)vs (2.4 ±1.2)].There was significant difference between the two groups in extubation quality score[(2.1 ±1.2)vs (3.2 ±1.4),P =0.0018].There was statistical difference between the two groups in T3 adrenal hormones[(N:120 ±25.3)pmol/L,(D:93.4 ±23.2)pmol/L,P =0.00].The dose of sufentanil (60.5 ±9.2)μg in group N was higher than (40.4 ±10.2)μg in group D (P =0.001).The dose of remifentanil (3.3 ±0.8)mg in group N was higher than (2.4 ±0.9)mg in group D (P =0.001).Conclusion Continuous pumped dexmedetomidine can effectively improve awakening quality of patients with double -lumen tube.

Objective To explore the effects of early nutritional support on immune function in postoperative patients with lung cancer. Methods Totally 84 cases of patients with lung cancer operation from May 2013 to May 2015 were selected and assigned to the control group and the observation group randomly, and 42 cases in each group. Patients in the control group were given routine nursing method, while patients in the observation group were given additional early nutritional support.Results Hospitalization time in the observation group was (9.73±2.18) d, while it in the control group was (12.52±3.46) (t=-4.42,P<0.01). 3 months after operation, the IgA (1.9±0.4) g/L, IgM(1.5±0.4) g/L, IgG (12.5±1.6) g/L and serum albumin (35.7± 5.2) g/L were all superior than that in the control group (t=2.59,2.90,3.96,2.81;P<0.05).Conclusions Early nutritional support is helpful in patients with lung cancer after chemotherapy for the restoration of nutritional and immune function, and it can improve the quality of life after operation.

Objective To observe the curative effects of applying laryngeal masks in the airway management in total laparoscopic hysterectomy in obese patients.Methods 60 cases of obese patients who underwent total laparoscopic hysterectomy were collected and were randomly divided into,the Endotracheal intubation (ET) group(n =30) and the laryngeal mask (LMA) group (n =30).The pre-operative co-existent diseases,applications of analgesia and intra-operative vital signs,the peak airway waves (Paw) 60min after pneumoperitoneum,blood gas;revival time;the incidences of complications were observed.Results 60min after pneumoperitoneum,MAP,HR,Paw and SpO2 values were:ET group:[(114.6 ± 22.4) mmHg,(108.2 ± 19.6) times/min,(25.4 ± 3.1) mmHg,(96.1 ± 1.2％)] LMA group:[(97.5 ±20.8)mmHg,(86.5 ±20.7) times/min,(22.6 ±3.7)mmHg,(98.9 ±0.8％)].The difference was statistically significant (P ＜ O.05);The blood gas analysis,PH,PaO2,PaCO2 and BE values are:ET group:[(7.34 ± 0.05),(177.5 ± 44.5) mmHg,(42.6 ± 6.1) mmHg,(-4.9 ± 0.8) mmol/L],LMA group:[(7.38 ± 0.05),(216.7 ± 46.6) mmHg,(38.4 ± 5.5) mmHg,(-3.1 ± 0.6) mmol/L].The difference was statistically significant (P ＜ 0.05);the status of complications:23 cases in ET group,accounting for 77％,10 cases in LMA group,accounting 30％,presenting significant differences (P ＜ 0.05).Conclusion The application of laryngeal masks in the airway management in total laparoscopic hysterectomy in obese patients facilitated the maintaining the stability of hemodynamics and blood gas,resulted in smooth post-anesthesia recovery and fewer complications.

Objective To observe the sedation and analgesia in surface anesthesia in conscious glossopha-ryngeal LMA for the clinical effect of laparoscopic hysterectomy.Methods 90 patients undergoing elective laparo-scopic total hysterectomy were randomly divided into three groups,30 cases in each group.The observation group (group A):sedation,analgesia,full surface anesthesia,control group 1 (B group)control group 2 (group C)were treated with endotracheal intubation under general anesthesia.At intubation and pull tube stage,the patients'reaction, hemodynamic changes,pneumoperitoneum 1 h blood gas and perioperative complications were observed.Results The mean arterial pressure(MAP)and heart rate(HR)of A group were (92.7 ±10.6)mmHg and (82.8 ±12.1)/min. Those of B group were (98.4 ±11.6)mmHg,(89.1 ±11.4)/min,C group were (111.2 ±12.5)mmHg,(104.1 ± 13.2)/min,those in A group and B group were better than C group(A group and C group,t =6.18,6.52,P <0.01;B group and C group,t =4.11,4.71,all P <0.01).The pulse oxygen saturation(SpO2 )and peak airway pressure (Paw)of A group were (99.6 ±0.4)and (17.3 ±2.1)mmHg,those in group B were (99.5 ±0.5)% and (17.6 ± 2.0)mmHg,group C were (99.5 ±0.5)% and (22.5 ±2.8)mmHg.The differences between A group and B group were statistically significant(compared with C group,t =14.3,8.14,all P <0.01;B group and C group,t =12.7, 7.78,all P <0.01).The incidence rates of perioperative complications and adverse reactions of A,B,C groups were 27%,33%,67%,that in C group was significantly higher than A group and B group(χ2 =9.64,6.67,all P <0.01). Conclusion The laryngeal mask airway was used for laparoscopic total hysterectomy under the condition of sedation and analgesia,and it can shorten the time of the whole body,prevent the difficulty of intubation,emergency airway and anesthesia related complications.