Erythrocytes, the most abundant cells in human body with excellent physiologic and morphologic characteristics, have been exploited extensively for their potential applications as drug delivering microspheres for several potential applications, including intravenous slow release of therapeutic agents, enzyme replacement therapy and drug targeting therapy. A series of methods has been tested successfully for the improvement of targeting to reticuloendothelial system.

Objective To study the pathogens associated with infectious keratitis for early diagnosis and treatment.Methods This study was conducted with the patients with confirmed diagnosis of infectious keratitis in our hospital during the period from January 2011 through December 2015.The specimens from corneal ulcer were subject to bacterial and fungal culture.The pathogens and associated risk factors of infectious keratitis were analyzed retrospectively.Results Of the 1 046 corneal specimens,369 (35.3％) were positive for microorganisms.The most frequently isolated microorganism was fungal species (53.1％),followed by grampositive cocci (31.2％),gram-negative bacilli (11.1％),and gram-positive bacilli (4.6％).Of the fungal strains,most were Fusarium (69.4％),followed by Aspergillus species (21.9％).Coagulase negative Staphylococcus accounted for more than half (50.4％) of the gram positive cocci.Conclusions Infectious keratitis is primarily associated with fungal infection,especially filamentous fungus such as Fusarium.

Objective To investigate the efficacy and safety of balloon-assisted catheter directed thrombolysis (CDT) for acute lower extremity deep vein thrombosis (DVT).Methods From September 2008 to February 2011,94 patients with acute lower extremity DVT were admitted.The cases in early stage were treated by CDT (Group A,n =50),and the cases in late stage were treated by balloon-assisted CDT ( Group B,n =44).The clinical data of these patients were retrospectively analyzed.The circumference difference between normal and affected limbs,scores of venous patency,and rates of venous patency were recorded for judging the efficacy.The total dose of urokinase and retention time of infusion catheter was compared between the two groups.The incidence of pulmonary embolism and bleeding were used to judge the safety of treatment.The venous patency was followed up by ultrasound or/and venography.Measurement data with normal distribution was described by mean + standard,and was analyzed using T test.Measurement data with non-normal distribution was described by M ( QL,QU ),QL =P25,QU =P75,and was analyzed using Wilcoxon' s test.Categorical variable data was analyzed using Chi-Square test Results The prior treatment circunfference difference between normal and affectéd limbs were (5.37 ±1.97) cm (thigh) & (4.14 ± 1.57) cm (calf) in Group A and (5.41±2.22) cm (thigh) & (4.05 ±1.61) cm (calf) in Group B ; and the difference between the groups was insignificant ( thigh:t =- 0.113,P=0.910; calf:t =0.288,P =0.774).The post treatment correspondences were:(2.96 ± 1.10) cm (thigh) & ( 1.93 ± 0.84 ) cm (calf) in Group A and ( 1.78 ± 1.40) cm ( thigh ) & ( 1.41± 1.17 ) cm (calf) in Group B; the difference between the groups was significant (thigh:t =4.66,P ＜0.0001; calf:t =2.548,P =0.012 ).The prior treatment score of venous patency was 9 (8,10) in Group A and 8.3(7,10) in Group B without significant difference (Z =- 1.5172,P =0.1292).The post treatment score of venous patency was 3.5 ( 2,5 ) in Group A and 0 ( 0,1) in Group B with significant difference ( Z =-5.7702,P ＜0.01).The rate of venous patency after the treatment was 55.0％ (42.3％,72.4％ ) in Group A and 100％ (88.5％,100％ ) in Group B,with significant difference ( Z =4.9148,P ＜ 0.01).The total dose of urokinase used in the treatment was 5.950 ( 5.525,7.225 ) × 106U in Group A and 4.100 (3.600,5.050) × 106U in Group B with significant difference (Z =-6.0133,P ＜ 0.01).The retention time of perfusion catheter was 10 (9,12) d in Group A and 6 (5,7) d in Group B with significant difference ( Z =- 8.0358,P ＜ 0.01).No symptomatic pulmonary embolism occurred in both groups during the treatment and follow-up period.The rate of bleeding complication was 38.0％ (19/50) in Group A and 22.3％ (10/44) in Group B,without significant difference (x2 =2.5590,P =0.1097 ).The removal rate of optional filter was 88.37％ (38/43) in Group A and 100％ (39/39) in Group B,with significant difference ( x2 =4.829,P =0.028 ).The rate of venous patency at the last follow-up point was 50.0％ (44.4％,59.2％ ) in Group A,and 95.4％ (83.6％,100％ ) in Group B,with significant difference (Z =- 3.2721,P =0.0011).Conclusions Balloon-assisted CDT was a promising treatment for acute lower-extremity DVT.It improved the effect of thrombolysis and reduced the dosage of urokinase,and did not increase the risk of pulmonary embolism.

Objective To evaluate the feasibility,safety and effectiveness of integrated treatmentwith comprehensively interventional techniques for acute deep venous thrombosis(DVT)of lower extremity. Methods During the period from June 2016 to June 2017,a total of 23 patients with acute DVT ofunilateral lower extremity were admitted to authors' hospital to receive integrated treatment. AngioJetthrombectomy,balloon angioplasty and iliac vein stent implantation were performed in the same setting.Angiography was performed immediately after the procedure. If the thrombus clearance rate was grade Ⅲand there were no large free clots(>5mm)in the lower extremity vein and inferior vena cava,the inferiorvena cava filters would be retrieved through their sheath,otherwise,the inferior vena cava filters would bereleased. All patients received continuous transcatheter infusion of urokinase until the thrombosis wasconfirmed to be completely dissolved. The patients were followed up at outpatient clinic at 1,3,6 and 12months after the treatment,and reexamination of color ultrasound and/or lower limb venography was used toassess the blood flow in the deep veins and in the stents. Results Angiography postoperatively showedthrombus clearance rate of grade Ⅲ was obtained in 20 patients(87%)who were successfully accomplishedintegrated treatment. The inferior vena cava filters had been retrieved after thrombolysis in 3 patients(13%)with thrombus clearance rate of grade Ⅱ. Twenty-one stents were implanted directly after thrombectomy in21 patients with iliac vein stenosis. There were no serious complications such as pulmonary embolism,severe hemorrhage,etc. Venous patency was confirmed by color ultrasound or lower limb venography. Conclusion Integrated treatment with comprehensively interventional techniques is feasible,safe,andeffective for acute DVT of lower extremity

Objective:To evaluate the association of microsatellite status with clinicopathological features of colorectal cancer patients after surgery.Methods:In total,572 colorectal cancer cases with clear pathological diagnosis and clinicopathological features from the Department of Pathology,Nanjing Drum Tower Hospital from June 2014 to June 2017 were included in the study.Microsatellite status and RAS mutations were detected by polymerase chain reaction.Ki67,EGFR,MGMT,and Lgr5 were detected by immunohistochemis-try.Clinicopathological features were analyzed based on the microsatellite states.Results:In this study,we found MSI-H in 7.0% of all patients,whereas 93.0% cases showed MSS/MSI-L.Compared with MSS and MSI-L colorectal cancer cases,MSI-H colorectal cancer cases showed the following characteristics:1)more likely located in the right colon,2)comparable incidences at different ages,3)ear-ly cancer stage(Ⅰ/Ⅱ)(P=0.003),4)a lower proportion of lymph node metastasis(P=0.023),5)a lower proportion of cancerous nod-ules(P=0.005),6)a lower mutation rate of KRAS(P=0.004),and no mutation of NRAS.There was no significant difference in the posi-tive rates of MGMT,EGFR,Lgr5,and Ki67 between the two groups.Conclusions:Compared with MSS/MSI-L patients,MSI-H patients have specific clinicopathological features.A lower mutation rate of KRAS/NRAS and higher methylation rate of MGMT were also found in MSI-H patients.Our results provide a basis for individual diagnosis and treatment in colorectal cancer.

Objective:To discuss the efficacy of pelvic lymph node dissection (PLND) on the patients undergoing radical cystectomy (RC).Methods:The clinicopathological data of bladder cancer patients who did not receive neoadjuvant chemotherapy and underwent RC in our center from November 2013 to December 2019 were collected. The average age of the patients was (67.4±10.9) years, including 284 males and 55 females. Postoperative pathology showed that 171 cases of MIBC and 168 cases of NMIBC. In the MIBC group, 124 patients received PLND. In the NMIBC group, 118 patients received PLND. There was no statistical difference of the PLND ratio between the two groups(72.5% vs. 65.5%, P=0.643). The average number of lymph nodes(LNs)in patients receiving PLND was(13.7±7.1). Explore the prognostic factors in NMIBC and MIBC respectively. The Kaplan-Meier method was used to analyze the effect of PLND on the prognosis of patients in MIBC and NMIBC group. Results:In MIBC group, 26 patients showed positive LNs, while 98 had negative LNs. 144 cases were high-grade urothelial carcinoma, and 47 cases received adjuvant treatment. In NMIBC group, 4 patients exhibited positive LNs, while 114 had negative LNs. 99 cases were high-grade urothelial carcinoma, and 15 cases received adjuvant treatment. After a median follow-up of 24(13, 43)months, Kaplan-Meier survival analysis showed that the 5-year overall survival(OS)of 395 patients was 63.6%. MIBC had a 5-year OS rate of 47.5%, while NMIBC had a rate of 79.1%. Univariate Cox regression showed that age≥65 years( HR=2.07, 95% CI 1.21-3.54, P=0.009), high tumor grade( HR=9.76, 95% CI 2.39-39.90, P<0.01), and positive lymph nodes( HR=2.47, 95% CI 1.27-4.78, P=0.008)were risk factors for the prognosis of MIBC.PLND ( HR=0.37, 95% CI 0.23-0.60, P<0.01) and adjuvant therapy ( HR=0.21, 95% CI 0.10-0.46, P<0.01) were protective factors of MIBC. However, the only risk factor of NMIBC was high tumor grade ( HR=6.66, 95% CI1.51-29.50, P=0.012). PLND had no effect on the prognosis of NMIBC patients following RC( HR=1.32, 95% CI 0.37-4.75, P=0.667). Multivariate COX regression analysis revealed that high tumor grade( HR=6.38, 95% CI 1.54-26.50, P=0.011) was independent risk factor of MIBC, PLND( HR=0.59, 95% CI 0.35-0.99, P=0.047), as well as adjuvant therapy ( HR=0.30, 95% CI 0.13-0.68, P=0.004) were independent protective factors of MIBC. Further analysis discovered that MIBC patients with negative LNs had a better prognosis than those with no PLND (62.4% vs. 16.1%, P<0.01)and positive LNs(62.4% vs.32.3%, P=0.005). However, there was no difference in prognosis between the negative LNs and no PLND group in NMIBC patients (81.3% vs. 66.6%, P=0.764). Conclusions:This study found that PLND was an independent predictive factor for MIBC patients receiving radical cystectomy.

Objective:To investigate the value of the vesical imaging reporting and data system score (VI-RADS) in the prognostic assessment of patients with bladder cancer.Methods:The data of 294 patients with pathologically confirmed bladder cancer in our department from February 2012 to September 2019 were retrospectively analyzed. Divide the patients into two groups based on the surgical method. In the transurethral resection of bladder tumor (TURBT) group, there were 121 cases, 102 males and 19 females; The average age of the patients was (66.7±12.3) years old, 52 cases <65 years old, 69 cases ≥65 years old, with VI-RADS <3 in 84 cases, VI-RADS ≥3 in 37 cases. In the radical cystectomy (RC) group, there were 173 cases, including 154 males and 19 females; The average age of the patients was (65.7±10.8) years, 77 cases <65 years old, 96 cases ≥65 years old, with VI-RADS <3 in 51 cases and VI-RADS ≥3 in 122 cases. The prognostic factors of TURBT group and RC group were analyzed, and the predictive value of VI-RADS score on overall survival (OS) and progression free survival (PFS) of bladder cancer patients after surgery was analyzed.Results:In this study, there were 294 cases with postoperative pathological diagnosis of urothelial carcinoma. The pathological staging was Ta stage in 104 cases (35.4%), T 1stage in 82 cases (27.9%), T 2 stage in 58 cases (19.7%), T 3 stage in 34 cases (11.6%), and T 4stage in 16 cases (5.4%). Pathological grading: 11 cases (3.7%) were low malignant potential, 77 cases (26.2%) were low grade, and 206 cases (70.1%) were high grade. There were 186 cases (63.3%) in the NMIBC group and 108 cases (36.7%) in the MIBC group. In the TURBT group, there were 114 cases (94.2%) in the NMIBC group and 7 cases (5.8%) in the MIBC group; In the RC group, there were 72 cases (41.6%) in the NMIBC group and 101 cases (58.4%) in the MIBC group. In the NMIBC group, the VI-RADS<3 and ≥3 were 128 cases (68.8%) and 58 cases (31.2%), respectively ( P<0.01); In the MIBC group, 101 cases (93.5%) had a VI-RADS <3 and 7 cases (6.5%) had a VI-RADS ≥ 3, respectively ( P<0.01). In the high grade of postoperative pathological group, 62 cases (30.1%) had a VI-RADS <3 and 144 cases (69.9%) had a VI-RADS ≥ 3, respectively ( P<0.01); In the non-high grade of postoperative pathological group, the VI-RADS <3 and ≥ 3 were 73 cases (83.0%) and 15 cases (17.0%), respectively ( P<0.01). The median OS survival for all patients in this study was 27.4 (16.6, 38.1)months and the median PFS survival was 24.7(14.0, 36.8) months. The results of univariate analysis showed that age ≥ 65 years old (OS: HR=6.09, P=0.001; PFS: HR=1.71, P=0.035), postoperative pathological diagnosis of tumor muscle infiltration (OS: HR=4.66, P<0.01; PFS: HR=2.24, P=0.001), postoperative high-grade tumor (OS: HR=4.26, P=0.008; PFS: HR=1.92, P=0.023), and VI-RADS score ≥ 3 (OS: HR=4.24, P=0.001; PFS: HR=2.21, P=0.002) were associated with poorer OS and PFS in patients. Multifactorial Cox model analysis revealed that a score of VI-RADS ≥3 was an independent risk factor for OS ( HR=3.41, P=0.012) and PFS ( HR=2.23, P=0.016). In the TURBT group, univariate analysis found that VI-RADS ≥3 ( HR=2.05, P=0.053) and high grade of postoperative pathology ( HR=2.77, P=0.005) were associated with poor PFS in patients, multifactorial Cox model analysis found only high grade of postoperative pathology ( HR=2.54, P=0.013) to be an independent risk factor for PFS. In the RC group, VI-RADS ≥3 ( HR=3.29, P=0.032) and age ≥65 years ( HR=5.37, P=0.001) were found to be independent risk factors for OS. The survival curve showed that the 5-year OS rates for groups with a VI-RADS ≥ 3 and <3 were 93.9% and 73.1%, respectively ( P<0.01), and the 5-year PFS rates for groups with a VI-RADS ≥ 3 and <3 were 76.5% and 53.0%, respectively ( P<0.01), with statistically significant differences. Conclusions:This study showed that VI-RADS ≥3 was an independent risk factor for prognosis in patients with bladder cancer and was more significant in patients receiving RC, but was not a significant predictor of prognosis in patients receiving TURBT.

Objective:To compare the prognosis and complications of muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC) patients undergoing radical cystectomy (RC) followed by ileal neobladder.Methods:The clinical data of 103 patients who underwent orthotopic ileal neobladder in Jiangsu Province Hospital from April 2010 to October 2021 were retrospectively analyzed. There were 51 MIBC patients and 52 NMIBC patients. In the MIBC group, there were 49 males and 2 females, aged (58.1 ± 8.9) years, with American Society of Anesthesiologists (ASA) score of 1-2 in 48 cases and 3 in 3 cases. Open radical cystectomy (ORC) was performed in 2 cases, laparoscopic (LRC) in 34 cases and robot-assisted radical cystectomy (RARC) in 15 cases. In the NMIBC group, there were 49 males and 3 females, aged (55.7 ± 9.9) years, ASA score of 1-2 in 51 cases and ASA score of 3 in 1 case. LRC was performed in 41 cases, and RARC in 11 cases. There were no statistically differences between the two groups in above indicators ( P>0.05). The Clavien-Dindo grading system (CCS) was used to assess the complications, defining CCS Ⅰ-Ⅱ as mild complications and CCS Ⅲ-Ⅴ as severe complications. According to their relationship to the neobladder, complications were be classified as neobladder-related and non-neobladder-related complications. The occurrence of complications and the prognosis of neobladder between MIBC and NMIBC were compared. Results:The average operation time of the MIBC group and NMIBC group were (421.2 ± 119.7) min vs. (439.8 ± 106.2) min. The blood loss were 400 (300, 700) ml vs. 400 (300, 625) ml. The frequency of lymph nodes removed were (14.9 ± 8.3) vs. (14.8 ± 8.5). The postoperative defecation time were 5 (4, 6) d vs. 5 (3, 6) d. And the postoperative hospital stay were 20 (15, 28) d vs. 22 (19, 28) d. There were no statistically differences between the two groups in above indicators ( P>0.05). The MIBC group had a significantly lower rate of pelvic lymph node metastasis [17.6% (9/51) vs. 0(0/52), P=0.001] and tumor thrombosis [23.5% (12/51) vs. 5.8% (3/51), P=0.011] than the MIBC group. Moreover, the NMIBC group had a considerably superior 5-year overall survival (OS) (97.6% vs. 70.2%, P=0.035). The proportion of pads needed in the daytime of the MIBC group and NMIBC group were 14.6% (7/46) vs. 6.7% (3/45). The frequency of urination were (2.0 ± 0.7) h vs. (2.4 ± 0.7) h. Furthermore, The proportion of pads needed at night were 47.9% (23/48) vs. 53.3% (24/45). The frequency of nocturnal urination were 3.1±1.5 vs. 2.3 ± 1.7. And the number of pads needed at night were all 1 (0, 1) pad. The daytime and nighttime incontinence rate were 25.0% (12/48) and 62.5% (30/48) respectively in MIBC, compared to 11.1% (5/45) and 62.2% (28/45) respectively in NMIBC. And the proportion of erectile function retention were 15.8% (6/38) vs. 25.0% (10/40). There were no statistically significant differences in the prognosis of neobladder function between the two groups ( P>0.05). Furthermore, the proportions of mild complications in the MIBC group and NMIBC group were [41.2% (21/51) vs. 51.9 (27/52)]. The proportions of severe complications were [21.6% (11/51) vs. 19.2% (27/52)]. The proportions of neobladder-related complications were [27.5% (14/51) vs. 25.0% (13/52)]. And the proportions of non-neobladder-related complications were [39.2% (20/51) vs. 25.0% (13/52)]. There were no statistically significant differences in the complications between the two groups ( P>0.05). Conclusions:There was no statistically significant difference in functional prognosis and complications of neobladder between MIBC group and NMIBC group, and NMIBC had a better oncologic prognosis.

Objective:To investigate the surgical efficacy and prognosis influencing factors of hilar cholangiocarcinoma based on multidisciplinary diagnosis and treatment.Methods:The retrospective cohort study was conducted. The clinicopathological data of 91 patients with hilar cholangiocarcinoma who underwent surgery in Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School from April 2004 to April 2021 were collected. There were 59 males and 32 females, aged (61±10)years. Patients who were admitted from April 2004 to March 2014 underwent traditional surgical diagnosis and treatment, and patients who were admitted from April 2014 to April 2021 underwent multidisciplinary diagnosis and treatment. Observation indica-tors: (1) surgical situations; (2) postoperative situations; (3) postoperative pathological examina-tions; (4) postoperative prognosis analysis; (5) influencing factors of postoperative prognosis. Follow-up was conducted using telephone interview and outpatient examination. Patients were followed up once every 6 months after surgery to detect survival. The follow-up was up to April 2023. Measure-ment data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the independent sample t test. Measurement data with skewed distribution were represented as M(range), and comparison between groups was conducted using the Mann-Whitney U test. Comparison of ordinal data was conducted using the rank sum test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher exact probability. The Kaplan-Meier method was used to draw survival curve and calculate survival rate. The Log-Rank test was used for survival analysis. Univariate and multivariate analyses were conducted using the COX proportional hazard model. Results:(1) Surgical situations. Of the 91 patients, there were 65 cases receiving hemi- or expanded hemi-hepatectomy, 13 cases receiving tri-hepatectomy, 9 cases receiving partial hepatectomy, 4 cases receiving extrahepatic bile duct resection. There were 24 cases receiving combined vein resection and reconstruction, 8 cases receiving combined pancreaticoduodenectomy, 6 cases receiving com-bined hepatic artery resection and reconstruction, including 24 cases receiving extended radical surgery (tri-hepatectomy, hepatic artery resection and reconstruction, hepatopancreaticoduodenec-tomy). The operation time, volume of intraoperative blood loss and intraoperative blood transfusion rate of 91 patients was (590±124)minutes, 800(range, 500?1 200)mL and 75.8%(69/91), respectively. Of the 91 patients, cases receiving extended radical surgery, the volume of intraoperative blood loss were 4, 650(range, 300?1 000)mL in the 31 patients who were admitted from April 2004 to March 2014, versus 20, 875 (range, 500?1 375)mL in the 60 patients who were admitted from April 2014 to April 2021, showing significant differences between them ( χ2=4.39, Z=0.31, P<0.05). (2) Post-operative situations. The postoperative duration of hospital stay and cases with postoperative infectious complications were (27±17)days and 50 in the 91 patients. Cases with abdominal infection, cases with infection of incision, cases with bacteremia and cases with pulmonary infection were 43, 7, 5, 8 in the 91 patients. One patient might have multiple infectious complications. Cases with bile leakage, cases with delayed gastric emptying, cases with chylous leakage, cases with liver failure, cases with pancreatic fistula, cases with intraperitoneal hemorrhage, cases with reoperation, cases dead during the postoperative 90 days were 30, 9, 9, 6, 5, 3, 6, 3 in the 91 patients. Cases with abdominal infection was 10 in the 31 patients who were admitted from April 2004 to March 2014, versus 33 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=4.24, P<0.05). Cases dead during the postoperative 90 days was 3 in the 31 patients who were admitted from April 2004 to March 2014, versus 0 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( P<0.05). (3) Post-operative pathological examinations. Of the 91 patients, cases with Bismuth type as type Ⅰ?Ⅱ, type Ⅲ, type Ⅳ, cases with T staging as Tis stage, T1 stage, T2a?2b stage, T3 stage, T4 stage, cases with N staging as N0 stage, N1 stage, N2 stage, cases with M staging as M0 stage, M1 stage, cases with TNM staging as 0 stage, Ⅰ stage, Ⅱ stage, Ⅲ stage, ⅣA stage, ⅣB stage, cases with R 0 radical resection, cases with R 1 or R 2 resection were 15, 46, 30, 1, 9, 25, 30, 26, 49, 36, 6, 85, 6, 1, 7, 13, 58, 6, 6, 63, 28. Cases with R 0 radical resection, cases with R 1 or R 2 resection were 15, 16 in the 31 patients who were admitted from April 2004 to March 2014, versus 48, 12 in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=9.59, P<0.05). (4) Postoperative prognosis analysis. Of the 91 patients, 3 cases who died within 90 days after surgery were excluded, and the 5-year overall survival rate and median overall survival time of the rest of 88 cases were 44.7% and 55 months. The 5-year overall survival rate was 33.5% in the 28 patients who were admitted from April 2004 to March 2014, versus 50.4% in the 60 patients who were admitted from April 2014 to April 2021, showing a significant difference between them ( χ2=5.31, P<0.05). Results of further analysis showed that the corresponding 5-year overall survival rate of cases without lymph node metastasis was 43.8% in the 16 patients who were admitted from April 2004 to March 2014, versus 61.6% in the 31 patients who were admitted from April 2014 to April 2021. There was a significant difference in the 5-year overall survival rate between these patients without lymph node metastasis ( χ2=3.98, P<0.05). The corresponding 5-year overall survival rate of cases with lymph node metastasis was 18.5% in the 12 patients who were admitted from April 2004 to March 2014, versus 37.7% in the 29 patients who were admitted from April 2014 to April 2021. There was no significant difference in the 5-year overall survival rate between these patients with lymph node metastasis ( χ2=2.25, P>0.05). (5) Influencing factors of postoperative prognosis. Results of multivariate analysis showed that poorly differentiated tumor and R 1 or R 2 resection were inde-pendent risk factors influencing prognosis after surgical treatment of hilar cholangiocarcinoma ( hazard ratio=2.62, 2.71, 95% confidence interval as 1.30?5.29, 1.30?5.69, P<0.05). Conclusions:Compared with traditional surgical diagnosis and treatment, treatment of hilar cholangiocarcinoma based on multidisciplinary diagnosis and treatment can expand surgical indications, reduce proportion of dead patients within 90 days after surgery, improve proportation of radical resection and long-term survival rate. Poorly differentiated tumor and R 1 or R 2 resection are independent risk factors influencing prognosis after surgical treatment of hilar cholangiocarcinoma.

Objective: To prepare human peripheral blood T lymphocytes with TIM3 (T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system. The transfection efficiency was examined 24 h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect hTIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of (41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0% (all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenic peptide;and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group (P<0.05). Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ (P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells (P<0.05). Conclusion: It’s simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.