ObjectiveTo construct human embryonic kidney cells (HEK293) modified with human preproenkephalin (hPPE) gene.MethodshPPE gene fragments were obtained from recombinant plasmid pcDNA3.1( + )/hPPE by using restriction endonuelease Hind Ⅲ and Not Ⅰ.Homologous recombination of lentivirus and hPPE gene was produced by using recombinant DNA technology.HEK293 cells were then transfected with the recombinant lentivirus vectors.The expression of hPPE gene in HEK293 cells was detected by Western blot.ResultsThe results of DNA sequencing indicated that the positive clone of recombinant lentivirus was completely consistent with sequencing of hPPE in Genebank.The titer of the concentrated virus was 2.07 × 108 TU/ml.GFP fluorescence was not seen in HEK293 cells transfected with the lentiviral vector under fluorescence microscope.A strong fluorescence was seen in HEK293 cells transfected with Ubc-GFP-L.V.empty viral vector.Positive expression of hPPE was demonstrated in HEK293 cells transfected with lentiviral vector by Western blot.Conclusion HEK293 cells modified with hPPE gene were successfully constructed and the target gene hPPE was stably expressed in HEK293 cells.

Trimethoprim molecularly imprinted polymer (MIP) was prepared as the coating of stir bar sorptive extraction (SBSE) and applied to the trace analysis of trimethoprim and three sulfonamides in complex samples.The MIP-coating was about 21.5 μm thickness with the relative standard deviations (RSD) of 5.9％ (n=10).It was homogeneous and dense with good thermal and chemical stability.The extraction capability of the MIP-coating was 1.7 times over that of the non imprinted polymer (NIP) coating.The MIP coating exhibited selective adsorption ability to sulfonamides,triazines and methotrexate besides antibacterial synergists.The methods for the determination of trimethoprim and three sulfonamides by MIP-coated stir bar sorptive extraction coupled with HPLC were developed.It was successfully applied to the trace trimethoprim analysis in spiked urine and plasma samples.The linear range was 5 to 200 μg/L and the detection limit was 1.6 μg/L.The recoveries in urine and plasma samples were 84.5％ to 91.7％ with RSDs of 2.9％ -4.4％,71.9％ to 85.1％ with RSDs of 3.0％ -7.3％,respectively.The trimethoprim MIP-coated stir bar was also applied to the trace sulfonamides analysis in spiked milk sample.The linear range was 10-200 μg/L,the detection limit was within the range of 4.5-6.1 μg/L,and the recovery was 83.2％ - 110.2％ with RSDs of 4.1％ -8.0％.

Objective To establish a tibial cancer pain model with MADB-106 mammary gland carcinoma cell line and to conduct therapeutic research through the behavior pain, X-ray, histological observation of the model. MethodsA rat model of bone cancer pain was established by intra-tibial inoculations of MADB-106 rat mamnary gland carcinoma cells in SD rats. Spontaneous pain was assessed by the reflection of spontaneous paw withdraw, move-evoked pain was observed by the extent of lower extremity claudication when the rats walked and heat hyperalgesia was evaluated by using a thermal dolorimeter. The structural damage of the bone was monitored by X-ray and histology.ResultsThe model group spontaneously withdrawed their paws (14.42±1.24) times on the 15th day after operation (P ＜0.001), (18.33±1.37) times on the 22nd day (P ＜0.001), (21.25±1.54) times on the 25th day (P ＜0.001). The radiant pain thresholds of the model group was (1 1.86±1.63) s on the 15th day after operation (P ＜0.001), (8.38±1.05) s on the 22nd day (P ＜0.001), (7.47±1.25) times on the 25th day (P ＜0.001). The move-evoked pain score of the model group was (1.25±0.62) on the 15th day after operation (P ＜0.001), (2.00±0.95) on the 22nd day (P ＜0.001), (2.33±1.07)on the 25th day (P ＜0.001). The results showed that rats of the model group displayed the gradual development of spontaneous pain, heat hyperalgesia and move-evoked pain on the 15-25 days after injection of the tumor cells. The X-ray of the tibia showed clear bone destruction. The histology of the bone showed the bone marrow cavity was full of tumor cells and the cortical bone had been destroyed. ConclusionThe bone cancer model has been built well and it will be useful to evaluate the therapy of cancer pain after two weeks.

Objective To summarize the evaluation standard of cardiac death organ donation from Chinese first category donor heart application and experience of donor heart function maintenance.Method From Jan.2013 to Jan.2015,donor hearts for heart transplantation were obtained in 18 cases of Chinese first category cardiac death organ donors from 109 cases of organ donation donors through rigorous assessment and effective donor heart function maintenance.The diagnosis of brain death was based on the diagnosis of brain death criteria (adult) by the neurological department of internal medicine,department of neurosurgery,and intensive care unit with brain death qualification.Organ donation work followed the basic principles of voluntary,free,fair,equitable and technical access.Result Eighteen case of heart transplantations were all operated with double lumen venous anastornosis.The cold ischemia time was (125.5+ 18.7) min (61-60 min),and cardiopulmonary bypass time was (130.4+ 12.5) min (99-193 min).In 18 heart transplantations,16 cases survived,and 2 eases died of acute right heart failure.During the follow-u period,the quality of life in the survival recipients was satisfactory.Conclusion Using cardiac death organ donation from Chinese first category donor heart transplantation has achieved good results.The accurate assessment of donor hearts and effective donor heart function maintenance are the key factors to guarantee the success of heart transplantation.

Objective To evaluate the release of leucine-enkephalin (L-EK) in the cerebrospinal fluid induced by intrathecal human embryonic kidney (HEK293) cells modified with human preproenkephalin (hPPE)gene and the analgesic efficacy of L-EK for bone cancer pain.Methods Forty CIBP female Sprague-Dawley rats were randomized into transplantation (CIBP+ hPPE/HEK293,n =20) and control (CIBP + HEK293,n =20)groups using a random number table.At 1 day before inoculation of cancer cells (T1,baseline) and 8,15,21,25,32 and 35 days after inoculation (T2-7),thermal paw withdrawal latency (TWL) was measured,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were recorded.After observation at T4,10 rats were chosen from each group and sacrificed and the cerebrospinal fluid of rats was collected in an ice bath for detection of hPPE expression using radioimmunoassay.Results Compared with control group,TWL was significantly prolonged,the concentration of L-EK in the cerebrospinal fluid was increased,and the number of licking/biting the claw on the transplantated side and degree of hindlimb limping during free activities were decreased at T4-7 in transplantation group.Conclusion Intrathecal HEK293 cells modified with hPPE gene can continuously secrete L-EK and mitigate bone cancer pain.

Objective To summarize the assessment standards of organ donation after cardiac death (DCD) donor lungs application and donor lung function maintenance experience.Method From Jan.2013 to June 2015,139 cases of DCD donors were subjected to rigorous assessment and effective donor lung function maintenance,and 11 donor lungs for lung transplantation were obtained.The donor lung cold ischemia time was (526.8-± 12.6) min (312 to 675 min).Double lung transplantation was performed on 9 cases,and 2 cases received single lung transplantation.Result Perioperatively,1 lung transplant recipient died of pulmonary infection.The survived 10 recipients had no rejection after operation,and obtained good quality of life during discharge to the final follow-up.Condusion The effect of donor lung transplantation using organ donation is satisfactory.The assessment standards and functional maintenance of donor lung are important factors to guarantee the success of lung transplantation.

Objective To investigate the effects of anti?PD?L1 monoclonal antibody and EGFR?TKI on expression of soluble PD?L1 and function of T lymphocytes in EGFR?mutated lung cancer cells. Methods Flow cytometry was used to analyze the expression of membrane PD?LI. ELISA was performed to detect the level of sPD?L1 in the supernatant of cultured EGFR?mutated and wild type lung cancer cells before and after erlotinib treatment. After treated with anti?PD?L1 monoclonal antibody alone and in combination with erlotinib, the proliferation of T lymphocytes in co?culture system was measured using Cell Counting Kit?8 ( CCK?8) assay. The expression levels of PD?LI and IFN?γin tumor cells and T lymphocytes treated with erlotinib in co?culture system were analyzed by flow cytometry and ELISA, respectively. Results PD?L1 was highly expressed in EGFR?mutated lung cancer PC9 cells (78.7±3.1)% and HCC827 cells (82. 7±2.6)%.After treated with erlotinib, the expression rates of membrane PD?L1 in PC9 and HCC827 cells were down?regulated (64.7%±3.1% and 73.0%±2.6%, respectively),significantly lower than that in the two cell lines without erlotinib treatment ( P<0.05) , and the expression levels of sPD?L1 in the supernatant of PC9 and HCC827 cells were also down?regulated ( 0. 680 ± 0. 120) ng/ml and ( 0. 903 ± 0. 047) ng/ml, respectively, significantly lower than that in the two cell lines without erlotinib treatment ( P<0. 01 ) . However, no significant changes of membrane PD?L1 and sPD?L1 expression were found in EGFR wild type lung cancer cells ( H1299 and A549) before and after erlotinib treatment. In the co?culture system composed of T cells and EGFR?mutated lung cancer cells, treatment with erlotinib alone promoted the proliferation of T lymphocytes (P<0.05), and combined treatment of anti?PD?L1 monoclonal antibody with erlotinib had a stronger effect ( P<0.05) . In the co?culture system composed of T cells and EGFR wild type cell lines, the proliferation of T cells was not changed after using erlotinib alone or combination of erlotinib and anti?PD?L1 monoclonal antibody ( P>0.05) . Before and after treatment with erlotinib, the secretion levels of IFN?γwere (856.0± 70. 3) pg/ml and ( 1 697. 3 ± 161. 0) pg/ml, respectively, showing a significant difference ( P<0.001). The expression rates of membrane PD?L1 were (76.2±0.5)% and (50.9±0.9)%, respectively, also showing a significant difference ( P<0.001) . However, no significant changes in the expression of IFN?γ and membrane PD?L1 were found in the co?culture system composed of T cells and A549 cells. Conclusions Anti?PD?L1 monoclonal antibody combined with EGFR?TKI can effectively promote the proliferation and secretion function of T lymphocytes in the microenvironment of EGFR?mutated lung cancer cells.

Objective To investigate the effects of anti?PD?L1 monoclonal antibody and EGFR?TKI on expression of soluble PD?L1 and function of T lymphocytes in EGFR?mutated lung cancer cells. Methods Flow cytometry was used to analyze the expression of membrane PD?LI. ELISA was performed to detect the level of sPD?L1 in the supernatant of cultured EGFR?mutated and wild type lung cancer cells before and after erlotinib treatment. After treated with anti?PD?L1 monoclonal antibody alone and in combination with erlotinib, the proliferation of T lymphocytes in co?culture system was measured using Cell Counting Kit?8 ( CCK?8) assay. The expression levels of PD?LI and IFN?γin tumor cells and T lymphocytes treated with erlotinib in co?culture system were analyzed by flow cytometry and ELISA, respectively. Results PD?L1 was highly expressed in EGFR?mutated lung cancer PC9 cells (78.7±3.1)% and HCC827 cells (82. 7±2.6)%.After treated with erlotinib, the expression rates of membrane PD?L1 in PC9 and HCC827 cells were down?regulated (64.7%±3.1% and 73.0%±2.6%, respectively),significantly lower than that in the two cell lines without erlotinib treatment ( P<0.05) , and the expression levels of sPD?L1 in the supernatant of PC9 and HCC827 cells were also down?regulated ( 0. 680 ± 0. 120) ng/ml and ( 0. 903 ± 0. 047) ng/ml, respectively, significantly lower than that in the two cell lines without erlotinib treatment ( P<0. 01 ) . However, no significant changes of membrane PD?L1 and sPD?L1 expression were found in EGFR wild type lung cancer cells ( H1299 and A549) before and after erlotinib treatment. In the co?culture system composed of T cells and EGFR?mutated lung cancer cells, treatment with erlotinib alone promoted the proliferation of T lymphocytes (P<0.05), and combined treatment of anti?PD?L1 monoclonal antibody with erlotinib had a stronger effect ( P<0.05) . In the co?culture system composed of T cells and EGFR wild type cell lines, the proliferation of T cells was not changed after using erlotinib alone or combination of erlotinib and anti?PD?L1 monoclonal antibody ( P>0.05) . Before and after treatment with erlotinib, the secretion levels of IFN?γwere (856.0± 70. 3) pg/ml and ( 1 697. 3 ± 161. 0) pg/ml, respectively, showing a significant difference ( P<0.001). The expression rates of membrane PD?L1 were (76.2±0.5)% and (50.9±0.9)%, respectively, also showing a significant difference ( P<0.001) . However, no significant changes in the expression of IFN?γ and membrane PD?L1 were found in the co?culture system composed of T cells and A549 cells. Conclusions Anti?PD?L1 monoclonal antibody combined with EGFR?TKI can effectively promote the proliferation and secretion function of T lymphocytes in the microenvironment of EGFR?mutated lung cancer cells.

Objective:To evaluate the influence of transcatheter aortic valve replacement (TAVR) on left atrial strain by two-dimensional speckle tracking echocardiography.Methods:Thirty-five patients, who were admitted for TAVR in Zhongshan Hospital of Fudan University from September 2015 to July 2018, were recruited. Echocardiography was performed 1 day before and 12 months after TAVR. Traditional ultrasound results, including aortic valve area (AVA), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular ejection fraction (LVEF), left atrial volume index (LAVI), peak velocity of tricuspid regurgitation (Vtr), peak velocity of the lateral wall of mitral annulus in early diastolic period (e′) and the ratios of peak mitral orifice velocity in early diastolic period to e′ (E/e′), were recorded. Two-dimensional speckle tracking imaging derived left atrial strain, which included reservoir (LASr), conduction (LAScd) and contraction (LASct), were recorded as well. The differences between pre-operation and post-operation were analyzed.Results:Compared to pre-operation, aortic valve area was increased ( P<0.001). Left ventricular systolic function was improved significantly (LVEDV and LVESV were decreased, LVEF was increased, all P<0.001). As to the left ventricular diastolic function, although LAVI and Vtr were decreased (both P<0.05), e′ and E/e′ were hardly changed (both P>0.05). Meanwhile, left atrial strain, including LASr, LAScd and LASct, were improved significantly 1 year post-TAVR (all P<0.01). Conclusions:Left atrial strain is able to evaluate the left atrial function of reservoir, conduction and contraction.Left atrial strain can be a promising tool of assessing left atrial function in patients underwent TAVR.

Objective:To observe the efficacy of vocal cord botulinum toxin type A injection in the treatment of refractory laryngeal contact granuloma and to analyze the factors affecting the curative effect.Methods:Fifty-two patients with refractory laryngeal contact granuloma who received vocal cord botulinum toxin type A injection under topical anesthesia from May 2021 to May 2023 were analyzed retrospectively. Among them, 51 were males and 1 was female, aged 22-66 (48.98±8.87)years old. All patients were followed up for a minimum of 12 months. Outcome measures in terms of patient cure rate, total effective rate, complications and recurrence rate were calculated. The median [ M ( Q1, Q3)] was used to represent non-normally distributed measurement data, and Logistic regression analysis was used to determine independent risk factors affecting efficacy. Results:The cure rate of 52 patients with refractory laryngeal contact granuloma treated by vocal cord botulinum toxin type A injection was 78.8% (41/52), and the total effective rate (including cure, marked and effective) was 90.4% (47/52). The median number of injections was 1[1,2]. Following a single injection, the cure rate was 69.2% (36/52), and the median treatment duration for cured patients was 3 [3,3] months. Hoarseness occurred in 88.5% (46/52) of patients, with recovery within 3 months in all cases. Additionally, 21.2% (11/52) of the patients experienced cough, sore throat, dyspnea, all of whom recovered within 3 months. One patient among the 41 cured cases was lost to follow-up, and the recurrence rate at the 12th month was 17.5% (7/40). Multivariate regression analysis indicated that age, granuloma size, history of PPI treatment, previous corticosteroid injections, prior surgical excision, pharyngeal reflux, chronic cough, pharyngeal reflux, chronic cough and vocal overuse were not independent risk factors for cure and recurrence.Conclusion:Vocal cord botulinum toxin type A injection is an alternative for the treatment of refractory laryngeal contact granuloma, which offers benefits such ashigh cure rate, short treatment cycle and less injection times.