ObjectiveTo study the effect and mechanism of Xiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine on immune escape in Lewis lung cancer mice. MethodA total of 60 specific-pathogen-free （SPF）-grade C57BL/6J male mice were injected subcutaneously with 0.2 mL of Lewis cell suspension （containing 2×10⁶ cells·mL^-1） in the right mid-axillary line. After 7 days， the mice that had been successfully modeled were randomly divided into six groups： the model group， the cisplatin group， the Xiangsha Liu Junzitang low-， medium-， and high-dose groups， and the combined group， with 10 mice in each group. The Xiangsha Liu Junzitang low-， medium- and high-dose groups were gavaged with 17.88， 35.75， 71.50 g·kg^-1 Xiangsha Liu Junzitang solution once a day， respectively， and the dosage of cisplatin intraperitoneally injected into the mice was converted to 5 mg·kg^-1 twice a week， and the tumour volumes of each group were measured every two days. The intervention lasted for 14 consecutive days. At the end of treatment， the tumour mass of mice in each group was weighed and the tumour inhibition rate was calculated. The morphological characteristics of tumours in each group were observed by hematoxylin-eosin （HE） staining. Fluorescent quantitative real-time polymerase chain reaction （Real-time PCR） assay was used to detect messenger ribonucleic acid （mRNA） contents of the natural killer group 2 member D （NKG2D） receptor， ribonucleic acid export-1 （RAE-1）， and γ interferon （IFN-γ） in the tumour tissues of each group. NKG2D， RAE-1， and IFN-γ mRNA in tumour tissues of each group. Immunohistochemistry （IHC） and Western blot were applied to detect the expressions of RAE-1， NKG2D， and IFN-γ in tumour tissues of each group， and Western blot was used to detect the expressions of interleukin-6 （IL-6）， Janus kinase 2 （JAK2）， p-JAK2， signal transducer and activator of transcription 3 （STAT3）， and p-STAT3 in tumour tissues of each group， as well as the protein levels of NKG2D， and RAE-1 in spleen tissues of each group. ResultCompared with that in the model group， the tumour mass decreased in all dose groups of Xiangsha Liu Junzitang， with no statistically significant difference. The tumour volume was reduced （P<0.05， P <0.01）. The pathological morphology was improved. The mRNA contents of NKG2D， RAE-1 and IFN-γ were increased in the medium-dose group （P<0.05， P<0.01）， and the protein expressions of NKG2D， RAE-1， and IFN-γ in tumour tissues were elevated （P<0.05， P<0.01）， and p-JAK2 and p-STAT3 protein expressions were decreased （P<0.05， P<0.01）. In spleen tissues， the protein expressions of NKG2D and RAE-1 in all dose groups of Xiangsha Liu Junzitang were significantly elevated （P<0.01）. Compared with those in the cisplatin group， NKG2D， RAE-1 and IFN-γ mRNA contents were elevated in the middle-dose group of Xiangsha Liu Junzitang， and the difference was not statistically significant. IHC showed that the protein expressions of NKG2D and IFN-γ in the combined group were significantly elevated （P<0.01）， and Western blot results showed that the protein expressions of RAE-1， NKG2D and IFN-γ were elevated （P<0.05， P<0.01）. p-JAK2 and p-STAT3 protein expressions were decreased in the combined group （P<0.05， P<0.01）. NKG2D and RAE-1 protein expressions were significantly increased in spleen tissues of the medium-dose groups and the combined group （P<0.01）. ConclusionXiangsha Liu Junzitang combined with phlegm-removing and detoxifying traditional Chinese medicine can inhibit the growth of tumours in Lewis lung cancer mice by up-regulating the expressions of RAE-1/NKG2D， promoting the activation of NK cells， and inhibiting immune escape， the mechanism of which may be related to down-regulation of the JAK2/STAT3 pathway.

Objective:To investigate the influence of liver drainage volume on overall survival time in patients with unresectable malignant hilar bile duct obstruction.Methods:Data of 633 patients with unresectable malignant hilar bile duct obstruction (BismuthⅡ-Ⅳ) who underwent endoscopic stent drainage in 3 endoscopy centers from January 2002 to May 2019 were retrospectively analyzed. Main observation indicators included clinical success rate, stent patency, overall survival, the effective liver drainage volume, and complication incidence.Results:The clinical success rates of patients with liver drainage volume <30%, 30%-50%, and >50% were 56.8% (25/44), 77.3% (201/260) and 84.2% (277/329) respectively. The incidences of early cholangitis were 31.8% (14/44), 18.8% (49/260) and 16.1% (53/329). The median stent patency time was 4.5 (95% CI: 1.8-7.2) months, 5.6 (95% CI: 5.0-6.2) months and 6.6 (95% CI: 5.2-8.0) months. The overall survival time was 2.4 (95% CI: 1.8-3.0) months, 4.0 (95% CI: 3.4-4.6) months and 4.9 (95% CI:4.4-5.4) months, respectively. The clinical success rate ( χ 2=8.28, P=0.012), median stent patency period ( χ 2=18.87, P=0.015) and overall survival time ( χ 2=6.93, P=0.024) of 30%-50% liver drainage volume group were significantly higher than those of <30% group. Further multivariate cox regression analysis showed that the disease type (hepatocellular carcinoma VS hilar cholangiocarcinoma: HR=1.50, 95% CI:1.18-1.91, P=0.001; gallbladder carcinoma VS hilar cholangiocarcinoma: HR=1.45, 95% CI:1.14-1.85, P=0.002; metastatic cholangiocarcinoma VS hilar cholangiocarcinoma: HR=1.48, 95% CI:1.08-2.04, P=0.015), bilirubin level >200 μmol/L ( HR=1.35, 95% CI:1.14-1.60, P<0.001),metal stents ( HR=0.67, 95% CI:0.56-0.79, P<0.001), liver drainage volume (volume 30%-50% VS <30%: HR=0.64, 95% CI: 0.45-0.90, P=0.010; volume>50% VS <30%: HR=0.58, 95% CI:0.41-0.81, P=0.002) and anti-tumor therapy ( HR=0.51, 95% CI:0.42-0.61, P<0.001) were independent predictors for overall survival time of patients with unresectable malignant hilar bile duct obstruction. Conclusion:When endoscopic stent drainage is performed for patients with unresectable malignant hilar bile duct obstruction, at least 30% liver volume is required for better overall survival. In addition, the use of metal stent drainage and anti-tumor therapy may increase survival benefits.

Objective:To explore the efficacy of the combination of radiofrequency ablation(RFA) and endoscopic metal stent in the treatment of patients with unresectable cholangiocarcinoma.Methods:From January 3, 2012 to June 30, 2019, at the Department of Endoscopic of the Third Affiliated Hospital of Naval Medical University, the clinical data of 44 patients with unresectable cholangiocarcinoma who were treated by the combination of RFA and endoscopic metal stent were retrospectively collected, which included age, gender, location of cholangiocarcinoma(hilar cholangiocarcinoma and distal cholangiocarcinoma), etc. Postoperative evaluation was conducted based on the follow-up, including clinical success rate, postoperative complication rate, time of stent patency and overall survival time (OS). The Kaplan-Meier method and log-rank test were used to analyze the difference of OS between patients with hilar cholangiocarcinoma and distal cholangiocarcinoma. Mann-Whitney U test was used for statistical analysis. Results:The age of the 44 patients with cholangiocarcinoma was (70.3±11.6) years old, with 20 males (45.5%). There were 22 patients (50.0%) with hilar cholangiocarcinoma and 22 patients (50.0%) with distal cholangiocarcinoma. The clinical success rate of 44 patients was 93.2%(41/44). A total of 5 patients(11.4%) had postoperative complications, which were all improved by appropriate treatment. The median time of follow-up of the 44 patient was 9.2 months(ranged from 3.1 to 57.6 months), the median time of stent patency was 7.0 months (ranged from 5.8 to 8.2 months). Thirty-two patients (72.7%) died during the follow-up, and the median OS was 10.9 months(ranged from 9.0 to 12.8 months). The median OS of patients with hilar cholangiocarcinoma was 7.8 months(ranged from 4.6 to 11.0 months) and that of patients with distal cholangiocarcinoma was 12.5 months(ranged from 5.7 to 19.4 months), and there was no statistically significant difference( P>0.05). Conclusion:RFA combined with endoscopic metal stent is safe and effective in the treatment of patients with unresectable cholangiocarcinoma.

Objective:To explore the long-term effect of endoscopic radiofrequency ablation for the treatment of unresectable ampullary carcinoma.Methods:Clinical data of patients with ampullary carcinoma who received endoscopic radiofrequency ablation in the Third Affiliated Hospital of Naval Medical University from January 2012 to May 2019 were retrospectively collected, including basic information, frequency of radiofrequency ablation, the type of biliary stent, postoperative complications, and follow-up. Kaplan-Meier method was used to analyze the survival of patients after endoscopic radiofrequency ablation. Relationship between frequency of radiofrequency ablation, type of biliary stent and overall survival time was analyzed.Results:A total of 50 patients were enrolled, including 31 males and 19 females, aged 73.0±9.7 years. Twenty-five patients (50.0%) underwent 1 radiofrequency ablation treatment, while 25 patients (50.0%) underwent radiofrequency ablation treatments more than twice. Postoperative complications occurred in 6 patients (12.0%), all of which were mild symptoms. The average follow-up was 22.3 months, with a total of 39 (78.0%) deaths, 5 (10.0%) lost to follow-up, and 6 (12.0%) surviving. The median overall survival time was 16.9 (95% CI: 9.1-24.8) months, with cumulative survival rates of 62.0%, 38.5%, 27.0%, and 12.6% at 1, 2, 3, and 5 years, respectively. The median overall survival time of those treated with radiofrequency ablation ≥2 times showed a trend of prolongation compared to patients treated once, but the difference was not statistically significant [26.7 (95% CI: 9.7-43.7) months VS 12.6 (95% CI: 4.9-20.3) months, χ2=3.049, P=0.081]. Plastic stents were used in 32 patients (64.0%) and metal stents in 18 patients (36.0%). There was no significant difference in median overall survival time between patients using metal and plastic stents [17.1 (95% CI: 6.1-28.0) months VS 15.9 (95% CI: 6.9-24.9) months， χ2=0.029， P=0.865]. Conclusion:Endoscopic radiofrequency ablation is a safe treatment for unresectable ampullary carcinoma, and multiple consecutive treatments may increase the survival benefit.

Objective:To evaluate the efficacy of endoscopic stenting drainage for patients with malignant hilar biliary obstruction caused by unresectable hepatocellular carcinoma.Methods:Data of 106 patients with malignant hilar obstruction caused by unresectable hepatocellular carcinoma who received endoscopic stenting drainage in the Third Affiliated Hospital of Naval Medical University from January 2012 to January 2019 were retrospectively analyzed. According to the different stent types, they were divided into the metal stent group (30 cases) and the plastic stent group (76 cases). The observation indexes included clinical success rate, complication incidence, stent patency and overall survival time. The independent predictors for stent patency and overall survival of patients were analyzed by multivariate Cox regression model.Results:The overall clinical success rate was 67.9% (72/106) and the incidence of postoperative cholangitis was 29.2% (31/106). The clinical success rates were 93.3% (28/30) and 57.9% (44/76) ( χ2=12.40, P<0.001), and the incidences of postoperative cholangitis were 13.3% (4/30) and 35.5% (27/76) ( χ2=5.12, P=0.024) in the metal stent group and the plastic stent group, respectively. The median stent patency was 5.2 (95% CI:3.7-6.0) months, and the overall survival time was 3.0 (95% CI:2.5-3.7) months. Multivariate Cox regression analysis showed that hepatic drainage volume <30% was an independent predictor for stent patency ( HR=2.12, 95% CI:1.01-4.46, P=0.048). Bismuth type Ⅳ ( HR=2.06, 95% CI:1.12-3.77, P=0.020), Child-Pugh class C ( HR=4.09, 95% CI: 2.00-8.39, P<0.001) and plastic stent ( HR=1.87, 95% CI:1.21-2.90, P=0.005) were independent predictors for overall survival time. Conclusion:Hepatocellular carcinoma involving the hilar bile duct has a poor prognosis. Compared with plastic stents, metal stents show advantages in clinical success rate and incidence of postoperative cholangitis. Hepatic drainage volume <30% is an independent predictor for stent patency, and Bismuth type Ⅳ, Child-Pugh class C and plastic stent are independent predictors for overall survival time.

Objective:To investigate the efficacy of endoscopic stent placement for patients with Bismuth type Ⅳ hilar cholangiocarcinoma.Methods:Data of 229 patients with unresectable Bismuth type Ⅳ hilar cholangiocarcinoma who successfully underwent endoscopic stent placement at the Department of Endoscopy, the Third Affiliated Hospital of Naval Medical University from January 2002 to January 2019 were retrospectively analyzed. Outcomes included clinical success rate, complication incidence, stent patency period and overall survival time. The patency of stents and overall survival time of patients were estimated by using the Kaplan-Meier method. The independent predictors for stent patency and overall survival of patients were analyzed by a multivariate Cox proportional regression model.Results:The overall clinical success rate was 78.2% (179/229). The incidence of early cholangitis after endoscopic retrograde cholangiopancreatography was 20.5% (47/229). The median stent patency and overall survival time were 5.7 (95% CI: 4.8-6.7) months and 5.1 (95% CI: 4.2-6.0) months, respectively. Further multivariate Cox regression analysis showed that metal stent ( P<0.001, HR=0.452, 95% CI: 0.307-0.666) and bilateral stents with bilateral angiography ( P=0.036, HR=0.644, 95% CI: 0.427-0.971) were independent predictors of stent patency; total bilirubin>200 μmol/L ( P=0.001, HR=1.627, 95% CI: 1.208-2.192), metal stent ( P=0.004, HR=0.636, 95% CI: 0.467-0.866) and antitumor therapy ( P<0.001, HR=0.439, 95% CI:0.308-0.626) were independent predictors of overall survival. Conclusion:There is high incidence of cholangitis in patients with unresectable Bismuth type Ⅳ hilar cholangiocarcinoma treated with endoscopic stenting. Longer stent patency can be achieved with metal stent placement and bilateral drainage. In addition, metal stent for drainage and antitumor therapy can also help increase the survival benefit.

CRISPR-Associated Protein 9 ; Gene Editing ; CRISPR-Cas Systems/genetics*

Objective:To explore the clinical characteristics of patients infected with 2019 novel coronavirus (2019-nCoV) Omicron variant in Zhuhai City.Methods:A retrospective study was conducted to compare the clinical characteristics of patients infected with 2019-nCoV Omicron variant (Omicron variant group, 39 cases) from January 13 to January 25, 2022, and those infected with 2019-nCoV non-Omicron variant (non-Omicron variant group, 98 cases) from January 17 to February 17, 2020, in the Fifth Affiliated Hospital of Sun Yat-sen University. Statistic methods were used by Mann-Whitney U test, chi-square test and Fisher exact probability test. Results:Among adults, the age of 21 patients in Omicron variant group was younger than that of 93 patients in non-Omicron variant group (34.0(26.0, 40.5) years vs 50.0(36.0, 62.0) years, Z=-3.81, P<0.001). Patients with underlying diseases in Omicron variant group were fewer than those in non-Omicron variant group (4.8%(1/21) vs 31.2%(29/93), χ2=6.17, P=0.013). The clinical classification of the Omicron variant group was mainly mild (71.4%(15/21)) or common type (28.6%(6/21)), while the non-Omicron variant group was dominated with common type (57.0%(53/93)) and severe type (23.7%(22/93)), and the clinical classification of the two groups had statistically difference ( χ2=31.79, P<0.001). The time of positive nucleic acid in Omicron variant group was longer than that in non-Omicron variant group (14.0(10.5, 16.5) d vs 8.5(4.0, 12.0) d, Z=-3.56, P<0.001). The proportion of pneumonia in Omicron variant group was less than that in non-Omicron variant group (28.6%(6/21) vs 86.0%(80/93), χ2=30.52, P<0.001). Differences were all statistically significant. There was no significant difference of the clinical characteristics in pediatric patients, but the proportion of children infected with 2019-nCoV Omicron variant was significantly higher than that infected with 2019-nCoV non-Omicron variant (46.2%(18/39) vs 5.1%(5/98), χ2=33.65, P<0.001). Conclusions:Adults infected with 2019-nCoV Omicron variant in Zhuhai City present with milder clinical symptoms, less proportion of pneumonia, but the time of positive nucleic acid is longer. The clinical characteristics of children infected with 2019-nCoV Omicron variant are not specific, while the proportion of children infected with 2019-nCoV Omicron variant is significantly higher.

Objective:To analyze the clinical characteristics of patients infected with 2019-nCoV Omicron variants BA.1 and BA.2 in Zhuhai city.Methods:A retrospective study was conducted to compare clinical characteristics of patients infected with 2019-nCoV Omicron variants BA.1 and BA.2, who were admitted in the Fifth Affiliated Hospital of Sun Yat-sen University during January 13 to March 8 2022. The Mann-Whitney U-test or Kruskal-Wallis H test was used for quantitative data, and the χ2 test or Fisher’s exact test was used for qualitative data. Results:Among 122 patients infected with the Omicron variant, there was 79 adults (BA.1 23 cases, BA.2 56 cases) and 43 children (BA.1 19 cases, BA.2 24 cases). In adults, patients infected with BA.2 sub-variant had a higher baseline viral loads at admission than BA.1 infected patients [7.64(6.92, 8.55) lg copies/mL vs. 6.64(6.04, 7.34) lg copies/mL; Z=-3.022, P=0.003]; compared to BA.1 patients, BA.2 patients had a higher proportion of mild and asymptomatic cases and a lower proportion of common infection cases ( χ2=8.052, P=0.012); the proportion of patients with pneumonia imaging changes in BA.1 patients was higher than that in BA.2 infected patients [(6/23, 26.1%) vs. (2/56, 3.6%); χ2=6.776, P=0.009). In children, the rate of fever in BA.2 group was higher than that in BA.1 group [(16/24, 66.7%) vs. ( 5/19, 26.3%); χ2=6.910, P=0.009); the proportion of patients with reduced lymphocyte counts in BA.2 group was higher than that in BA.1 group [(17/24, 70.8%) vs.(1/19, 5.3%); χ2=18.734, P<0.001). Compared with adult cases, children with BA.2 sub-variant infection had higher fever rate [(16/24, 66.7%) vs. (19/56, 33.9%); χ2=7.317, P=0.007). The viral loads of daily nasal swabs in BA.2 infected patients increased first and then decreased in both adults and children, with a greater decrease than BA.1 during the first two weeks. Conclusions:Compare with 2019-nCoV Omicron variant BA.1, BA.2 has a higher baseline viral loads in adults, which means much more contagious in the early stages. But the viral load drops faster in BA.2 infected patients. In children, BA.2 patients are more likely to have fever and reduced lymphocyte counts, which indicates that the prevention and control of 2019-nCoV Omicron sub-variant BA.2 is more difficult.

Objective:To evaluate the feasibility, efficacy, and safety of ultra-fine metal stents for malignant hilar biliary strictures (MHBS).Methods:Ultra-fine metal stents were simultaneously placed into the intrahepatic duct of 30 patients with unresectable malignant hilar biliary strictures of Bismuth type Ⅱ or higher (based on imaging or histological and/or cytological findings) by using the stent-by-stent mode from January 2014 to June 2021 at the Department of Gastroenterology, Eastern Hepatobiliary Surgical Hospital. The technical success rate, clinical efficacy and incidence of complications were observed.Results:The technical success rate was 100.0% (30/30), and the clinical success was achieved in 28 patients (93.3%) . The mean procedure time was 55.7±20.7 min and the placement time was 28.3±18.2 min. Early adverse events included mild acute pancreatitis ( n=2) and cholangitis ( n=5). The median stent patency and the median overall survival were 243 days (95% CI: 186.6-299.4 days) and 237 days (95% CI: 149.0- 325.0 days), respectively. The incidence of late cholangitis was 36.7% (11/30). Stent malfunction occurred in 14 of the 30 patients, and 5 of them received endoscopic reintervention. The technical success rate for the reintervention was 4/5 with the mean procedure time of 49.8±6.9 min. Conclusion:Malignant hilar biliary obstruction endoscopic palliation with ultra-fine metal stents using the stent-by-stent mode is safe, feasible and effective.