Objective To observe the in vivo gene expression profile of the recombinant adenoassociated-2 virus mediated human GM-CSF, mouse GM-CSF (rAAV-2-hGM-CSF, rAAV-2-mGM-CSE )vector modified bone marrow mesenchymal stem cell (BMSC). Methods We transduced the BMSC by rAAV-2-hGM-CSF, rAAV-2-mGM-CSF at the condition which have acquired before respectively, then transfused the in vitro gene modified BMSC after 12 days proliferation in vitro to 6 weeks old nude mice through tail vein,while the BMSC transfused in control group hadn' t been gene modified. 2, 4, 6, 8 weeks after transfusion, count the total white blood cells and detect the hGM-CSF, mGM-CSF concentration in nude mice serum at that time point. Results Nude mice serum hGM-CSF levels were 23.77, 25.32, 19.77, 15.25 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 36.25 ng/L, the in vitro level before transfusion; mGM-CSF levels were 34.96, 34.84, 35.50, 32.93 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 25.14 ng/L, the in vitro level before transfusion; at the same time point the nude mice serum mGM-CSF levels were 17.34,17.44, 14.68, 16.85 ng/L in control group, rAAV-2-mGM-CSF transduced BMSC made the nude mice white blood cell count increased, but no changes in nude mice white blood cell count at rAAV-2-hGM-CSFtransduced BMSC and control group. Conclusion BMSC as a gene therapy vehicle, it can be gene modified in vitro, then the gene modified BMSC could let the therapeutic gene to have therapeutic effects in vivo.

Objective　To evaluate the diagnosis and surgical treatment of hilar cholangiocarcinoma(H-CC). Methods　Retrospective analysis was made on the clinical feature, surgical treatment and the effect on 73 patients with H-CC. Results　Diagnosis was made in all of the patients preoperatively and the correct diagnostic rate of BUS was 69.9%. In the treatment, radical resection was performed on 15 patients with good results in a short-term period. Of the 43 patients who underwent biliary tract internal drainage or exterrnal drainage, 37 patients had good results in a short-term period, while 6 died after operation. Laparotomy or hepatic artery cannulization with chemotherapy was performed on 15 patients and no change occurred in a short-term period after operation. In 15 cases subjected to radical resection, 11 cases were followed up. The 1,3-year survival rates was 90.9%, 20.0% respectively, but none of the patients survived for over 5 years. In patients undergoing other operations, none survived more than 9 months. Conclusions　It's still difficult to mak early diagnosis of H-CC, which mainly depends on imaging technics. The BUS should be choiced first. Radical resection rate is still low nowadays. The lobus quadratus resection is helpful to select the operation.

A sensitive colorimetric method for the detection of copper ions (Cu2+) was developed based on the surface modification of silver/platinum nanoclusters (Ag/Pt NCs) and regulation of peroxidase-like activity. It was found that 3-mercaptopropionic acid (MPA) could inhibit the catalytic ability of Ag/Pt NCs; however, it lost the inhibition toward catalytic ability of Ag/Pt NCs after oxidized by oxygen through the catalysis of Cu2+. On the basis of this, a colorimetric method was developed for the detection of Cu2+ through measuring the colorimetric signal variation of the TMB-H2O2 reaction. This method exhibited high sensitivity and selectivity toward Cu2+ over a panel of other metal ions. The linear range was 10-100 nmol/L and the detection limit was 5.0 nmol/L (3σ). The above method was also applied to detect real water samples and spiked samples, and the results demonstrated that this method was simple with low cost.

Objective To evaluate the efficacy of Flu/CTX conditioning regimen for the treatment of severe aplastic anemia in pa- tients receiving allogeneic hematopoietic stem cell transplantation. Methods Nine patients with severe aplastic anemia received HLA identi- cal peripheral blood hematopoietic stem cell transplantation (PBSCT) using Flu/CTX conditioning regimen, which consisted of fludarbine [30 mg/(m2 d) for5 days (-7 to -3) ], CTX [50mg/(kg d) for4 days(-5 to-2)]. All patients received cyclosporin A (CsA) and mycophenolet mofetil (MMF) for prophylaxis of graft-versus-host disease(GVHD). Results The Fiu/CTX regimen was very well toler- ated, with no severe regimen related toxicity. In all patients, the median days of neutrephil exceeding 0. 5×109/L and platelet exceeding 20 ×109/L were 12 days (range 10-16 days) and 16 days (range 14-19 days), respectively. Complete chimerism was achieved in all pa- tients at one month after PBSCT. Two patients had acute GVHD and one had chronic GVHD. In the 39-month median follow-up duration, all patients were alive in disease-free situation. Conclusion The Flu/CTX conditioning regimen may reduce transplantation-related toxicities and can achieve full chimerism and high long-term disease-free survival. Allogeneic hematopoietic stem cell transplantation using intravenous Fiu/CTX conditioning regimen is a safe and effective treatment method for the patients with severe aplastic anemia.

BACKGROUND: Recombinant adeno-associated virus 2(rAAV-2) has attracted considerable attention due to its nonpathogenic nature in contrast to other viral vectors such as adenoviral and retroviral vectors in gene therapy attempts.OBJECTIVE: To explore rAAV-2 transduction to bone marrow mesenchymalstem cell(BMSC) in vitro and evaluate the possibility of using rAAV-2 as a vector for gene therapy of acute myelogenous leukemia(AML).DESIGN: An open experiment with cells as the observational subjects.SETTING: Department of Hematology, Nanfang Hospital, Southern Medical University.MATERIALS: The experiment was conducted in the Department of Hematology, Nanfang Hospital, Southern Medical University from February to July 2004. We used passages 3 to 5 BMSCs derived from six de novo AML patients and four healthy volunteers in this study.METHODS: BMSC was isolated from 6 to 10 mL of bone marrow aspirates obtained from the iliac crests of the patients who had been diagnosed as having de novo AML and from those of healthy volunteers. The acquired BMSC was infected by rAAV-2 which contained enhanced green fluorescent protein (rAAV-2-eGFP) at different multiplicity of infection(MOI) (MOI = 1 × 102,1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107) . Then we observed through phase contrast fluorescent microscope and flow cytometer to evaluate green fluorescent protein(GFP) expression 10 to 14 days after transduction. GFP expression was observed as the rAAV-2-eGFP transduced BMSC cultured in vitro. We also observed the in vitro gene expression profile of GFP in rAAV-2-eGFP transduced BMSC which was selected by neomycin ( G418). First, we confirmed GFP expression in BMSC through phase contrast fluorescent microscope, then on flow cytometer to detect the percentage of GFP expression.MAIN OUTCOME MEASURES: The efficiency of rAAV-2-eGFP transduction to BMSC. GFP expression was observed through phase contrast fluorescent microscope and flow cytometer at different time points after transduction.rAAV-2-eGFP to BMSC derived from normal volunteers and AML patients had no significant differences. GFP began to express 10 to 14 days after transduction, and the transduction efficiency ranged from 0. 3% to 1.4%. By changing infection condition, we could not make a higher transduction efficiency( P ＞ 0.05) . One round infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was ( 1. 030 ± 0. 034) %, 3 rounds of infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was (1. 140 ±0. 036)%, and coinfected by LipofectAMINE was (1. 380 ± 0. 054)%. However, 293 cell line which was the package cell of rAAV-2 could be efficiently transduced by AML patients transduced by rAAV-2-eGFP at MOI = 1 × 105: The percentage of GFP expression cell gradually decreased from 1.14% at day 12 after transduction to 0. 6% as cell passaged from 2 to 3, and maintained at a level of 0. 5% to 0. 6% later on till 61 days after transduction. After selected by neomycin(G418) 1 month later, rAAV-2-eGFP transduced BMSCs could maintain a long-term GFP expression at a level of 6.0% in vitro without significant decay within 100 days of observation period after transduction.CONCLUSION: The advantages of rAAV-2 mediated gene transduction lie in safety, no immune response to the host, and long-term expression maintained by the target gene. rAAV-2 and BMSC can be used for in vitro gene therapy, and as a systemic gene delivery system, it might be an alternative for systemic gene therapy in the future.

Objective To evaluate the efficacy and feasibility of Flu/ivBu/Tl" conditioning regimen for the treatment of refractory or relapsed acute non-lymphocytic leukemia in patients receiving allogeneic hematopoietie stem cell transplantation. Methods Seven patients with refractory or relapsed acute non-lymphocytic leukemia received HLA identical peripheral blood hematopoietie stem cell transplantation (PBSCT) following Flu/ivBu/TY conditioning regimen, which consisted of fludarbine, busulfex and thiotepa. All patients received cyclos-porin A (CsA) and mycophenolet mofetil (MMF) for prophylaxis of graft - versus - host disease (GVHD). Results The Flu/IVBu/TT regimen was tolerated very well, without severe regimen related toxicity. In the 31-month median follow-up duration, 5 of 7 patients were a-live in disease-free situation. Conclusion The Flu/ivBu/TT conditioning regimen reduced transplantation-related toxicities and offered high long-term disease-free survival, and was tolerated very well. Allogeneie hematopoietie stem cell transplantation using Flu/ivBu/TT condition-ing regimen is a safe and effective option for the patients with refractory/relapsed acute non-lymphocytic leukemia.

OBJECTIVETo perform genotyping analysis and subsequent prenatal genetic diagnosis for two families affected with oculocutaneous albinism (OCA).

METHODSDirect sequencing of TYR and P genes was performed in two albino probands. Family members were screened for corresponding mutant alleles. Prenatal genetic diagnoses were performed at early pregnancy by chorionic villus sampling (CVS) at mid-pregnancy through amniocentesis.

RESULTSNo mutations were detected in the TYR gene in either probands, whereas 4 heterozygous mutations of the P gene were found, namely c.406C>T, c.535A>G, c.808-2A>G and c.2180T>C, among which c.535A>G and c.808-2A>G were novel. In the first round prenatal genetic testing, both fetuses were found to have the same genotypes as the probands. Both families had decided to terminate the pregnancy after genetic counseling. In the second round testing, neither of the fetuses was found to be affected by genotyping. The pregnancies continued and two healthy fetuses were born.

CONCLUSIONOCA can be classified by genotyping, with which reliable prenatal diagnosis and feasible genetic counseling may be provided.

Adolescent ; Adult ; Albinism, Oculocutaneous ; diagnosis ; embryology ; enzymology ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Child ; Child, Preschool ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genotype ; Humans ; Infant ; Male ; Membrane Transport Proteins ; genetics ; Middle Aged ; Molecular Sequence Data ; Monophenol Monooxygenase ; genetics ; Pedigree ; Point Mutation ; Pregnancy ; Prenatal Diagnosis ; Young Adult

Immune deficiency of the host caused by allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the initial factor of reactivation of latent human cytomegalovirus (HCMV). The risk factors of reactivation of HCMV in allo-HSCT recipients consist of the serological status of HCMV in donors and recipients, the matching degree of human leukocyte antigen (HLA) and pretreatment patterns, etc. The reactivation of HCMV is associated with the expression of a series of viral cleavage and proliferation proteins induced by the overexpression of major immediate early promoter/enhancer (MIEP) in the viral genome. In this article, the risk factors of reactivation of HCMV after allo-HSCT, the molecular changes related to maintaining latent infection of HCMV, the key role of MIEP overexpression in reactivation of HCMV, and the molecular pathways involved in reactivation of HCMV after allo-HSCT were reviewed and the major molecular events of reactivation of HCMV after allo-HSCT were elucidated, aiming to provide reference for the prevention and treatment of cytomegaloviral disease (CMVD) after allo-HSCT.

Objective To investigate the correlation between disability acceptance and personality trait in patients with cerebral infarction, so as to provide a new perspective to improve the disability acceptance. Methods A total of 205 patients with cerebral infarction were investigated by the Acceptance of Disability Scale and the Eysenck Personality Questionnaire-revised-Short Scale for Chinese( EPQ-RSC) . The level of personality trait in patients was compared with the norm, and the Pearson correlation analysis and hierarchical regression analysis were used to explore the effect of personality trait on the disability acceptance. Results The total score of disability acceptance was (128. 08 ± 28. 89) in patients, and 57. 07% of them (117/205) were in the low level, 27. 80% of them (57/205) were in the medium level, 15. 13% of them (31/205) were in the high level. The level of neuroticism, psychoticism and lie in personality trait were significantly higher than those of the norm (t=4. 143,12. 622, 6. 604, respectively;P<0. 01). The level of introvert or extrovert character in personality trait was significantly lower than that in the normal population (t= -9. 552,P<0. 01). Pearson correlation analysis showed that each factor of disability acceptance was significantly negatively correlated with the scores of neuroticism, psychoticism and dissimulation in personality trait (r= -0. 436 0. 025,P<0. 05), but significantly positively correlated with the score of introvert or extrovert character in personality trait ( P<0. 05). Hierarchical regression analysis showed that dissimulation, introvert or extrovert character and neuroticism in personality trait could explained 30. 50% of the total variance of disability acceptance in patients with cerebral infarction (P <0. 05). Conclusions The level of disability acceptance is in the low level in cerebral infarction patients, and personality trait is the influencing factor. Nursing staff can start from the personality trait to improve the level of disability acceptance in cerebral infarction patients.

Objective:To evaluate the efficacy and safety of ultrasound-guided high intensity focused ultrasound(HIFU)on pain intensity,pain sensation and overall survival in patients with advanced pancreatic cancer. Methods:Clinical data of advanced pancreatic cancer patients treated by HIFU were collected from the patients enrolled during August 2020 to September 2022 at the second department for oncology of Yueyang Hospital of Integrated Chinese and Western Medicine affiliated to Shanghai University of Traditional Chinese Medicine.In this study,SPSS 26.0 software was used for the statistical analysis of NRS score and BPI score.The Kaplan-Meier survival analysis method was applied to calculate the median overall survival(OS)and then the survival curve was drawn.At the same time,the incidence of related adverse reactions during and after HIFU treatment was counted. Results:(1)Among the 45 patients,30 patients received HIFU combined with chemotherapy,and the other 15 patients only received HIFU.(2)Among the 45 patients,32 patients had pain relief after HIFU treatment,and the NRS score kept decreased across 1 week,2 weeks,3 weeks and 1 month after HIFU treatment(P＜0.05).The pain sensation score of BPI scale also decreased correspondingly,and the difference was statistically significant(P＜0.05).(3)The median OS of 45 patients was 11.1 months(95％Cl:9.30-1 2.90),of which 30 patients treated with HIFU combined chemotherapy had a median OS of 12.4 months(95％Cl:9.1 8-15.62),and 15 patients treated with HIFU only had a median OS of 4.6 months(95％Cl:1.11-8.10).(4)No serious adverse events were observed in all patients during and after HIFU treatment.Only 5 patients had asymptomatic mild elevation of blood amylase,and the incidence of mild adverse reactions was 11.1％. Conclusion:HIFU can effectively relieve pain and prolong the median survival time in patients with advanced pancreatic cancer.