Aplastic anemia(AA)is a rare disease oringed from hematopoietic cell,characterized by failure of the bone marrow and pancytopenia in peripheral blood.The mechanism of AA is indistinct,it can be congenital,or acquired disposition.Acquired AA may be secondary to poisonous,radiation,virus infection and some medicine.All these factors can injure bone marrow directly,suppressing the proliferation and differentiation of bone marrow,on the other hand,the immune system play a pivotal role indirectly.This review focused on the pathologic classification,mechanism of drug-induced aplastic anemia.

BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA interference detected with real-time fluorescence quantitative RT-PCR and Western blot.RESULTS: Sequence analysis showed that GFAP-shRNA recombinant plasmid eukaryotic expression vector was successfully constructed, and optimal GFAP-shRNA eukaryotic vector was screened using real-time fluorescence quantitative RT-PCR and Western blot. The GFAP expressions were reduced by 81%, 63% and 56% at the levels of mRNA and protein respectively.CONCLUSION: GFAP-shRNA eukaryotic expression vectors were successfully constructed and screened. The gene expression GFAP of primitive rat astrocyte can be suppressed significantly by the GFAP-shRNA eukaryotic expression recombinant optimized via ex vivo cellular expression suppression model, which should pave the way for the following multiple targets of RNAi genetic manipulation in the treatment of suppression of glial scar formation after spinal cord injury.

OBJECTIVE:To introduce the Fair PharmaCare program of BC Canada for the reference of the reform of China's medical insurance system.METHODS:The running procedure of Fair PharmaCare program of BC Canada was introduced in detail and its characteristics were analyzed.RESULTS & CONCLUSIONS:Fair PharmaCare program covers all BC residents and protects them against economic difficulties in pharmacare.The program serves as a reference for the reform of China's medical insurance system.

Now the in-service staff have been permitted to apply for master's degree with the same educational level aspostgraduates. This is a signal reforming policy in postgraduate education and degrees management area in China. Manyhigher professionals are trained for our socialist modernization construction by this education technology. Although therelatively perfect system to manage this project was established in China, there are still some problems demandingprompt solution on the management thoughts, management process as well as management methods in some authorizedunits.

Objective To investigate the distribution of complications and the relationship with neurologic subtype and gross motor function in preterm infants with cerebral palsy (CP). Methods The type, grade of Gross Motor Function Classification System (GMFCS), intelligence,speech, ophthalmologic consultation, brainstem auditory evoked potential and electroencephalogram of 135 preterm infants with CP were reviewed. Results There were 284 complications in total, (2.10±1.33) per child, and was significantly different among various types of CP (F=5.50, P<0.001). The incidence of mental retardation and speech disorder was significant different among various types (P<0.05). The incidence of mental retardation, speech disorder, visual impairment and epilepsy increased significantly (P<0.05) in spastic quadriplegia infants,compared with those with diplegia and hemiplegia. The incidence of mental retardation, speech disorder, visual impairment and musculoskeletal disorder was significantly different (P<0.05) among various grades of GMFCS. The frequency of complications was more in children unable to walk (GMFCS Ⅳ~Ⅴ) than able to walk (GMFCS Ⅰ~Ⅲ) for children over 2 years old (t=70.05, P<0.001). Conclusion The incidence of mental retardation, speech disorder, visual and hearing impairment, secondary musculoskeletal disorder and the multiple disorders are related with neurologic subtype and/or the grade of GMFCS.

Objective To evaluate the selectively killing effect of adenovirus(Ad)mediated double suicide gene driven by VEGF promoter on pancreatic cancer cell SW1990. Methods VEGFexpressing SW1990 were infected by Ad-VEGFP-CDTK and Ad-null.respectively.The infection rate was observed and the expression of CDTK was detected by RT-PCR and Western blotting.Followed by treatment with 5-FC and GCV killing effects were evaluated and bystander effects were analyzed by MTF.Pathological character of cells was observed by electron microscopy and distribution of cell cycle was detected by flow cytometry.The caspase-3 activity was detected by absorption spectrometry. Results The infection rate of the resultant recombinant Ad to SW1990 cells was not apparently different.RT-PCR and Western blotting demonstrated product of CDTK gene in SW1990 cell infected by Ad-VEGFP-CDTK.Prodrug could inhibit proliferation of SW1990 and the effect was dose-dependent.There was considerable bystander effect as observed by MTF.Apoptotic peak was also shown by flow cytometry.Morphologic features of apoptosis in SW1990 cells were displayed via electron microscopy.Cells at the G0-G1 phase was increasing and the rate at the G2-M and S phase was decreased by prodrug.The caspase-3 activity gradually rised with the increasing concentration of the prodrug. Conclusions The CDTK fusion gene system controlled by VEGF promoter has killing effect on the VEGF-expressing SW1990 cells and inducing the cell apoptosis.

Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.

BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.

Objective To evaluate the efficacy of three-dimensional conformal radiotherapy (3DCRT) combined with concurrent chemotherapy for loco-regionally recurrent or mastastatie rectal cancer. Methods Between June 2004 and January 2007,47 patients with loco-regionally recurrent or nmatastatic rectal cancer were treated by 3DCRT of 55-65 Gy in 1.8-2.0 Gy fractions. Chemotherapy was given concurrenfly using oxaliplatin(100 mg/m2 ,iv drop,d1 ) and capecitabine(1500 mg/m2,orally,dl-14,21 days per cycle). Results After the follow-up of 12-35 months, the total response rate, complete response rate and partial response rate were 79% (37/47) ,19% (9/47) and 60% (28/47) ,respectively. The pain-alleviation rate and the mean pain-alleviation time were 85% and 6 months. The 1- and 2-year survival rates were 83% and 51%. Quality of life was improved without any treatment related death. Conclusions 3DCRT combined with concurrent chemotherapy is effective and well-tolerated in patients with post-operatively locoregionally recurrent or mastastatic rectal cancer.