Bradykinin (BK) is a calcium/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) specific activator, and Cyclosporin A (CSA) is reported to suppress protein phosphotase (PP)-2B activity. In vitro studies have shown that CaMKⅡ and PP-2B play an important role in Alzheimer-like phosphorylation of microtube-associated protein tau. To reconstitute an animal model based on the imbalance of protein kinase (s) and protein phosphatase (s) seen in Alzheimer brain, we injected BK and/or CSA into rat hippocampus. The results from behavioral study showed that an obvious disturbance in learning and memory was seen with BK or BK plus CSA injected rats. Moreover, the behavior abnormality appeared earlier in aged rats than young adults of the same kind after the injection. On the other hand, no obvious dysfunction in living and behavior was observed with CSA alone injected rats. The results obtained by immunohistochemical assay indicated that the staining for M4\, 12E8\, PHF-1 and CaMKⅡ was stronger, and for Tau-1 was weaker in BK injected rats compared with Control group. It was also found that the binding of M4 and PHF-1 but not 12E8 to tau was significantly increased in CSA injected rats. As the same as BK injection, binding of Tau-1 to tau was decreased after CSA injection. The immunostaining for 12E8\,PHF-1 and CaMKⅡ was increased, whereas for Tau-1\, M4\, and GSK-3 was decreased after combination injection of BK and CSA. In addition, the staining of PP-2B decreased in all the three models. To our knowledge, this is the first data shown in vivo that the activation of CaMKⅡ induces both Alzheimer-like tau phosphorylation and behavioral disturbance.

Objective To reconstitute an animal model based on the imbalance of protein kinase(s) and protein phosphatase(s) seen in Alzheimer brain. Methods The injection of bradykinin (BK) into hipocampus was performed, and their behaviors were observed by electronic attack-jump experiment and phosphorylation of tau using immunohistochemical assay. Results The results of behavior studying showed that an obvious disturbance in learning and memory was seen in BK injected rats. The results obtained by immunohistochemical assay indicated that the staining for M4?12E8?PHF-1 and CaMK-Ⅱ was stronger, and for tau-1 was weaker in BK injected rats as compared with the control group. The BK injected rats showed obvious deficit in behavior [mistakes made by model and control rats during electronic attack-jump experiment:8.3?2.5 and 6.9?3.1, P

Ca 2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) is a member of a family of Ca 2+/calmodulin-regulated protein kinases which also includes Ca 2+/calmodulin-dependent protein kinases Ⅰ and Ⅲ, myosin light chain kinases and phosphorylase kinase. Unlike the other members of this family, CaMKⅡ is multifunctional protein kinase and distributes in a variety of tissues. It is especially abundant in neuronal system. In hippocampus, CaMKⅡ is about 2% of the total protein. Studies have shown that CaMKⅡ plays an important role in a variety of biological processes, such as regulation of gene transcription, synthesis of neurotransmitter, phosphorylation of cytoskeletonal protein, hippocampal learning and memory formation.

Obiective:To establish a new and better model to investigate the properties of mast cells that couldbe involved in acupuncture process mechanisms.Methods:Connective tissue under the conum at the area of acupuncture point Zusanli(ST 36)from rat was acutely bluntly separated with forceps and scissors。And incubated in bath solution up to several hours.Mast cells in slices of that tissue were irradiated with laser light of 650 mn,and changes in the appearance were observed under mlcroscope. In addition.patch.clamp technique in whole-cell configuration was employed to induce mechano-sensitive currents by pressure applied through the patch pipette.Results:1)A high density of mast cells embedded in the extracellular matrix was detected in the tissue slices using toluidine blue staining.The mast cells survived for up to several hours;2)Laser irradiation for 10 min(40 mW) resulted in fusion of intracellular vesicles with the plasma membrane of the mast-cell surface;3)Whole.cell currents increased when pressure gradients of-30 cm,-60 cm or-90 cm H20 were applied,the response was attenuated in the presence of 1 0 u M Ruthenium Red(a common blocker of channels 0f the vallinoid.sensitive transient receptor potential family TRPV).Conclusion:The skin tissue slice of rat Zusanli(ST 36)may be used as a model to investigate the role of mast cells in acupuncture.t he results obtained in this model support the suggestion that skin mast cells play a role In laser as well as mechanical acupuncture and TRPV channels may be involved in acupuncture effects.

OBJECTIVETo reconstitute an Alzheimer's disease model by administering bradykinin (BK) or cyclosporine A (CSA) to the rat hippocampus.

METHODSBK or CSA was administered to the rat hippocampus using a stereotaxic apparatus. The behavior of the rats was observed with an electronic attack jump platform. The phosphorylation of Tau protein was examined through immunohistochemical assay.

RESULTSBehavior studies showed that an obvious disturbance in learning and memory was seen in BK injected rats.No obvious dysfunction was observed in CSA injected rats. The results obtained by immunohistochemical assay indicated that the staining of M4, 12E8, paired helical filament-1 (PHF-1) and calcium/calmodulin-dependent protein kinase II (CaMKII) was stronger, and that of Tau-1 was weaker in BK injected rats compared with the control group. We also found that the binding of M4 and PHF-1 but not 12E8 to Tau was significantly increased in CSA injected rats. As for BK injection, binding of Tau-1 to Tau was decreased after CSA injection.

CONCLUSIONTo our knowledge, this is the first data showing in vivo that the activation of CaMKII induces both Alzheimer-like Tau phosphorylation and behavioral disturbances.

Alzheimer Disease ; etiology ; Animals ; Bradykinin ; toxicity ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; metabolism ; Cyclosporine ; toxicity ; Disease Models, Animal ; Hippocampus ; drug effects ; metabolism ; Immunohistochemistry ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; tau Proteins ; analysis ; metabolism

Objective: To explore the clinical efficacies of applying aged marginal donor liver. Methods: From January 2015 to June 2018, clinical data were retrospectively analyzed for 199 adult liver transplantation donors and recipients. They were divided into two groups of aged (≥60 years) and appropriate age (<60 years). The prognosis of two groups was compared after a follow-up period of 1 year. And the aged group was further assigned into lower and higher fat infiltration groups according to the degree of fat infiltration in donor liver and compared the prognosis of two groups. Results: No significant differences existed in initial, peak value and recovery time of transaminase (AST/ALT), peak value and recovery time of total bilirubin, glutamyl transpeptidase, alkaline phosphatase, international normalized ratio (INR), peak value of lactate, postoperative hospital stay, graft dysfunction, biliary/vascular complications, acute/chronic rejection or graft survival rate between aged and appropriate age groups post-transplantation. The aged group was further divided into lower and higher fat infiltration groups according to the fat infiltration rate (<20%, ≥20%). And significant inter-group differences existed in peak value and recovery time of AST/ALT, peak value of total bilirubin, glutamyl transpeptidase, lactate, postoperative hospital stay and graft dysfunction. The above parameters were significantly worse in higher fat infiltration group. Also the rejection rate was higher in high group at 1 year post-operation and no significant inter-group difference existed in biliary/vascular complications. In higher group, 4 patients showed graft dysfunctions during perioperative period. Two of them were discharged successfully after secondary transplantation and another 2 patients died. Conclusions On the premise of comprehensive evaluations of donor liver status and reasonable matching of recipients, aged marginal donor liver can be safely applied with excellent clinical outcomes. Severe fatty donor liver should be employed with caution. Hypertensive drugs, high serum sodium and long period of cold ischemia are also important influencing factors for aged donors.

Objective:To explore the effect of pre-transplant immunotherapy on the prognosis of transplant recipients with hepatocellular carcinoma(HCC).Methods:From June 2018 to September 2021, retrospective analysis was conducted for clinical data of 19 HCC-liver transplant recipients receiving pre-transplant immunotherapy in affiliated Huashan Hospital of Fudan University. Pre-transplant immunotherapy regimen, adverse reactions, post-transplant acute rejection, tumor recurrence and metastasis and other complications were recorded. According to the preoperative tumor imaging and the changes of alpha-fetoprotein level, tumor change during recipient waiting period was judged by the mRECIST standard. According to whether or not there was partial tumor remission, they were divided into two groups of non-remission( n=13)and remission( n=6). Postoperative conditions of two groups were compared. Kaplan-Meier method was used for calculating the survival rate of recipients after transplantation and survival curve and Log-rank test utilized for comparing the recurrence-free and overall survival rates of recipients at 1 and 2 years post-operation. Results:A total of 19 liver transplant recipients received immunotherapy plus targeted and transcatheter arterial chemoembolization(TACE) before transplant. In non-remission group, tumor was stable( n=9)and progressive( n=4); 6 cases in remission group had tumor partial remission. Two recipients in non-remission group were pathologically confirmed by liver biopsy to have acute rejection(2/19, 10.5%)and both recovered after glucocorticoid + rATG and glucocorticoid therapy. In non-remission group, 2 patients died from septic shock post-operation. Among 3 patients of tumor recurrence and metastasis post-operation, 2 cases survived with tumor and 1 died after tumor recurrence and metastasis. In remission group( n=6), none had postoperative tumor recurrence and metastasis. The recurrence-free survival rates of non-remission group recipients at 1 and 2 years post-operation were 76.9% and 76.9% and recurrence-free survival rates in remission group were 100% and 100% respectively and inter-group difference in RFS was not statistically significant( χ2=1.468, P=0.226). The overall survival rates of recipients in non-remission group at 1 and 2 years post-operation were 76.9% and 76.9% respectively. And recipients in remission group were 100% and 100% respectively and no statistically significant inter-group difference existed in OS( χ2=1.292, P=0.256). Conclusions:Without a significantly higher risk of acute rejection after transplant, immunotherapy may be an effective option for bridging treatment before liver transplantation for HCC. And it remains necessary to expand the sample size for verifications and supports.

Objective:To explore the relationship between CD24 expression in preoperative peripheral blood as well as cancer tissue and clinical parameters and prognosis in patients with hepatocellular carcinoma (HCC) after liver transplantation (LT).Methods:From November 2018 to November 2019, clinical data were collected for 65 HCC patients and 41 patients with benign liver disease.The preoperative peripheral blood level of CD24 was detected by enzyme-linked immunosorbent assay (ELISA) and the expression of CD24 in cancerous foci and adjacent tissues examined by immunohistochemistry.Kaplan-Meier survival curves of differential CD24 expression were plotted and survival differences compared by Log-rank method.One-way ANOVA was utilized for examining the relationship between the expression level of CD24 and various clinicopathological parameters and multivariate Cox analysis for screening independent risk factors affecting patient prognosis.Results:The concentration of CD24 in preoperative peripheral blood (p-CD24) of HCC patients (6.51±2.33 μg/L) was significantly higher than that of patients with benign liver disease (4.10±0.91) μg/L, P<0.05.The positive rate of CD24 was obviously higher in cancerous tissues than that in adjacent tissues (87.7% vs. 4.6%, P<0.05). The peripheral blood level of CD24 was positively correlated with the expression intensity of CD24 in tumor tissues (t-CD24, r=0.570, P<0.001). The expression of CD24 (both in blood and cancer foci) was significantly correlated with preoperative level of gamma-glutamyl transferase (GGT), maximal tumor diameter, microvascular invasion, portal vein tumor thrombus, vessel carcinoma embolus and satellite focus ( P<0.05). The expression of CD24 in patients exceeding the Milan/UCSF criteria was higher than those fulfilling the criteria ( P<0.005). Patients with a higher expression of CD24 had worse overall survival and recurrence-free survival rates as compared to those a lower expression of CD24 ( P<0.05). Multivariate Cox analysis indicated that t-CD24 [OS: HR=3.661(1.005-13.333)], P=0.049; recurrence-free survival (RFS): [HR=4.331(1.887-9.942), P=0.001] and preoperative level of alpha fetoprotein (AFP) [OS: HR=4.900(1.590-15.097), P=0.006]; RFS: [HR=3.414(1.614-7.221), P=0.001] were independent risk factors for overall survival and recurrence-free survival in HCC patients undergoing LT. Conclusions:The preoperative peripheral blood level of CD24 in HCC patients undergoing LT indirectly reflects the expression of CD24 in cancerous tissues to a certain extent.And the expression of CD24 in cancerous tissue is one of the independent risk factors affecting OS and RFS of LT patients.

Objective To explore the clinical features and risk factors associated with intrahepatic and hilar cholangiocarcinoma after liver transplantation .Methods Retrospective analysis of clinical data was performed for 20 hospitalized patients with intrahepatic and hilar cholangiocarcinoma from June 25 ,2014 to October 31 ,2018 .Treatments and follow-up outcomes were analyzed .The survival rate was calculated by the Kaplan-Meier method and the survival curve plotted .Cox regression model was employed for analyzing the prognostic factors .Results The cumulative recurrence rate of patients with AJCC stage Ⅰ /Ⅱ was significantly lower than that in AJCC stage Ⅲ/Ⅳ .And the cumulative recurrence rate of stageⅠ/Ⅱ Patients was 0 and that of stage Ⅲ/Ⅳ 76％ (P=0 .042) .Cox regression model showed that CA19-9 was the only prognostic factor .An elevated level of CA19-9 was associated with high recurrence post-transplantation (HR=1 .001;95％ CI:1 .000～1 .001;P=0 .035) .Conclusions During progressive stage ,the recurrence rate is higher with a worse prognosis .And an elevation of CA19-9 is an independent poor prognostic factor after intrahepatic and hilar cholangiocarcinoma transplantation .

Objective To investigate the clinical efficacy and therapeutic progress of orthotopic liver transplantation for the treatment of hepatic epithelioid hemangioendotheliom(a EHE). Methods Clinical data of 2 patients diagnosed with hepatic EHE were retrospectively analyzed. One patien(t case 1) was diagnosed with multiple hepatic EHE complicated with multiple infarction lesions of the spleen, and underwent orthotopic liver transplantation combined with splenectomy. The other cas(e case 2) was diagnosed with multiple hepatic EHE and received orthotopci liver transplantation alone. Literature review was performed. Pathological characteristics, clinical efficacy of liver transplantation and clinical prognosis of hepatic EHE patients were analyzed. Results Two patients successfully underwent surgery and were discharged postoperatively. The diagnosis of hepatic EHE was confirmed by pathological examinaiton and case 1 was complicated with EHE of the spleen . For case 1, tacrolimus was replaced by sirolimus at postoperavtie 1 month. At postoperative 4 months, capecitabine was orall y administered( chemotherapy) for EHE recurrence. At 6 months after surgery, the patient wa sdiagnosed with recurrent hepatic EHE complicated with multiple bone metastases, and waso rally administered with sorafenib. At postoperative 7 months, the patient died from cachexia and liver failure. Case 2 was followed up until the submission date( 8 months after surgery). No postoperative complications and tumor recurrence were observed. Previous studies had demonstrated that surgical resection was the primary therapy of hepatic EHE. Liver transplantation was highly recommended for patients with multiple unresectable hepatic EHE and extra-hepatic lesions. Moreover, chemotherapy, percutaneous puncture combined with transcatheter arterial chemoembolization and anti-angiogenesis treatment exerted certain clinical efficacy.C onclusions Surgical resection remains the primary therapy of hepatic EHE. For patients with multiple intrahepatic EHE, liver transplantation is considered as the optimal treatment. Much attention should be diverted to the prevention and treatment o f recurrent hepatic EHE following liver transplantation, aiming to improve the clinical efficacy.