Objective:To express the recombinant single chain Fv(scFv) in E.coli and reduce immunogenicity and molecular weight of a monoclonal antibody specific for human platelet.Methods:The variable regions of the heavy and light chains of platelet specific antibody SZ 2 were amplified by reverse transcription and polymerase chain reaction.VH and VL gene segments were cloned into pUC Tm and joined together with a (gly 4ser) 3 linker.The resulting scFv was expressed in PET expression system.The expressed recombinant protein was characterized by its size on SDS PAGE,by Western blot,by flow cytometry and its functions.Results:The VH and VL genes were homologous with the published gene sequences of mouse antibody variable region.The recombinant scFv was expressed mostly in the form of inclusion bodies,and the yield was up to 25% of the total cell proteins.Functional studies showed that SZ 2 scFv could bind to platelet and could suppress platelet aggregation induced by ristocatin and thrombin.Conclusion:A recombinant SZ 2 scFv specific against platelet was developed and characterized.

Purpose　The aim is to obtain the cDNA sequence of encoding extramembrane human FL gene with high level expression in E.coli. Methods　The primers were designed based on the known FL cDNA sequence. The total RNA was isolated from fetal liver cells , and then RT-PCR was performed. The fragment was cloned into pUC-18T vector, and further sequenced by automatic sequence analyzer. The gene was inserted into GST fusion expression vector between BamH Ⅰ and EcoR Ⅰ sites. The recombinant plasmid was transformed into E.coli strain DH5 α and induced with 1mmol/L IPTG.Results　The 546bp DNA fragment was amplified by RT-PCR method from fetal liver cells and its sequence was identical to the published sequence encoding human FL. The expressed fusion protein, with molecular weight of about 22kD, was about 10% of the total bacteria protein by SDS-PAGE and densitometry analysis.Conclusion　cDNA was cloned successfully. This study provided a basis for the further fundamental research and clinical application of FL.

AIM: To further investagate the mechanism of thrombus formation and develop a new remedy of anti-thrombus formation. METHODS: The amplified DNA fragment of vWF-A1 domain was inserted into expression vector with 6?his taq (pQE-31), the recombinant expression vect or was transformed into E coli (strain M15) and induced by IPTG. The recombinant fragment, comprising residues 449-728 of mature vWF subunit, designate rvWF-A1. It was purified by Ni-NTA agarose column and renatured by Tris buffer containin g GSH and GSSG. FACS and platelet aggregometer were employed to analyse the rvWF -A1 function of binding to platelet glycoprotein Ib and inhibiting ristocetin-in duced platelet aggregation. RESULTS: The rvWF-A1 was expressed successfully in E coli, comin g up to 30% of total bacterial protein. Its purify was over 95% through Ni-NTA a garose. It was identified to have ability to bind to GPIb, its biologic activity to inhibit ristocetin-induced platelet aggregation was observed, and the inhibi tive rate was 84 7%. CONCLUSION: The above results indicated that high-level expressi on of rvWF-A1 was successfully achieved in E coli and rvWF-A1 may be an effectiv e antithromotic agent in preventing thrombus formation.

Objective To observe the protective and regeneration-promoting effects of Ruyi Zhenbao Pill (RZP) on nerve injury in zebrafish.Methods The zebrafish model of central nervous injury was induced by mycophenolate mofetil,the model of peripheral motor nerve and axonal injury was induced by ethanol,and the model of myelin damage was induced by ethidium bromide.The variations of central nerve,axon and myelin sheath fluorescence intensity,and peripheral motor nerve length in zebrafish,which exposed to the different concentration (10.0,33.3,and 100.0 μ.g/mL) of RZP,were observed with fluorescence microscope.The effective protection rates of RZP on zebrafish central nerve and axone,and the regeneration-promoting effect on peripheral motor nerve and myelin sheath were analyzed and calculated with the image processing software NIDS-Element's.Results In 10.0,33.3,and 100.0 μg/mL RZP groups,the zebrafish central nervous injury protective rates were 2％,24％,and 50％ (P ＜ 0.001),respectively,the peripheral nerve regeneration promoting rates were 44％ (P ＜ 0.05),49％ (P ＜ 0.01),and 93 ％ (P ＜ 0.001),the axonal injury recovery rates were 3％,29％ and 48％ (P ＜ 0.05),and the myelin sheath regeneration promoting rates were 36％ (P ＜ 0.01),37％ (P ＜ 0.001),and 41％ (P ＜ 0.001),compared with model group.Conclusions RZP could not only protect the central nervous and axonal injury,but also promote the regeneration of peripheral nerve and myelin sheath in zebrafish.

AIM:Atherosclerotic calcification is highly linked with plaque instability and cardiovascular events .Adenosine monophosphate-activated protein kinase ( AMPK) has been involved in the pathogenesis of various cardiovascular disease .The contributions of AMPKαsubunits to the development of atherosclerotic calcification in vivo remained unknown .We hypothesized that AMPKαsubunits may play a role in the development of atherosclerotic calcification .METHODS: Atherosclerotic calcification was generated by 24-week fed of western diet in ApoE-/-background mice .Calcification was evaluated in aortic roots and innominate arteries of ApoE-/-mice or in mice with dual deficiencies of ApoE and AMPKαsubunits globally ( AMPKα1 and AMPKα2 ) , or vascular smooth muscle cell ( VSMC)-specific or macrophage-specific knockout of AMPKα1 with atherosclerotic calcification pone diet . The mechanism of AMPKα1 in regulating Runx2 was further explored in human aortic VSMC .RESULTS: Ablation of AMPKα1 but not AMPKα2 in ApoE-/-background promoted atherosclerotic calcification with increased Runt -related transcription factor ( Runx2 ) expression in VSMC compared with ApoE-/-mice.Conversely, chronic administration of metformin, which activated AMPK, markedly reduced ath-erosclerotic calcification and Runx2 expression in ApoE-/-mice but had less effects in ApoE-/-/AMPKα1 -/-mice.Furthermore, VSMC-but not macrophage-specific deficiency of AMPKα1 in ApoE-/-background promoted atherosclerotic calcification in vivo com-pared with the controls .AMPKα1 silencing in human aortic VSMC prevented Runx 2 from proteasome degradation to trigger osteoblastic differentiation of VSMC .Conversely , activation of AMPK led to Runx 2 instability by inducing its small ubiquitin-like modifier modifi-cation (SUMOylation).Protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, was directly phosphorylated by
AMPKα1 at serine 510, to enhance its SUMO E3-ligase activity.Ablation of PIAS1 serine 510 phosphorylation inhibited metformin-in-duced Runx2 SUMOylation, and subsequently prevented the effect of metformin on reducing oxLDL-triggered Runx2 expression in hu-man aortic VSMC.CONCLUSION:Deficiency of AMPKα1 in VSMC increases Runx2 expression and promotes atherosclerotic calcifi-cation in vivo.AMPKα1 phosphorylates PIAS1 to enhance Runx2 SUMOyalation and subsequent degradation .

In this article, the Chinese Traditional Medicine and Materia Medica Subject Headings, Standards of the People's Republic of China - Classification and Codes of Diseases and Zheng of Traditional Chinese Medicine and the Chinese Terms in Traditional Chinese Medicine and Pharmacy were compared. Three standards were compared from the terminology quantity, content and classification. Each standard has its special feature. The compatibility and consistency are not strong in these standards. More authoritative traditional Chinese medicine terminology standards need to be established for the application in the clinical practice and scientific research.

Objective To investigate the cytogenetic and clinical features of acute myeloid leukemia (AML) with CD7 positive. Methods Among 788 AML patients in the First Affiliated Hospital of USTC from January 2008 to December 2012, a total of 140 AML patients with CD7 positive were enrolled, and their clinical and cytogenetic characteristics were analyzed respectively. Results According to French-American-British (FAB) classification systems, M5[47.1 % (66/140)] and M2[27.1 % (38/140)] were often detected in 140 AML patients with CD7 positive. The positive rate of CD7 in M0patients [(60.9±13.2) %] was the highest, followed by (53.1±29.5) % in M1patient. Karyotype analysis showed that 72 (51.4 %) AML patients with CD7 positive had unfavorable karyotypes. Thirty-one (22.1 %) AML patients with CD7 positive simultaneously showed the expressions of lymphoid antigens. Clinically, some AML patients with CD7 positive was accompanied by hyperleukocytosis [75.0 % (105/140)] (white blood count ≥20×109/L) and hepatosplenomegaly [82.1 % (115/140)]. The proportion of elder patients (above 65 years old) and complete remission rate of AML with CD7 positive were lower than those of AML with CD7 negative [25.7 % (36/140) vs. 39.4 % (255/648);12.1 % (17/140) vs. 24.7 % (160/648), respectively], and there were statistical differences (χ 2＝ 8.62, P＝0.03; χ 2＝ 9.70, P＝ 0.01, respectively). Conclusion AML patients with CD7 positive have specific cytogenetic and clinical characteristics, and poor prognosis.

Objective:To explore the relationship between the reconstitution of immune cells in patients with hematological malignancies and the occurrence of chronic graft-versus-host disease (cGVHD) after treatment with unrelated cord blood transplantation (UCBT) and sibling peripheral blood stem cell transplantation (PBSCT) .Methods:A total of 124 patients undergoing allogenic hematopoietic stem cell transplantation (allo-HSCT) in the First Affiliated Hospital of University of Science and Technology of China from March 2018 to August 2019, including 96 patients with UCBT and 28 patients with PBSCT. Peripheral blood immune cells of patients with UCBT and PBSCT were detected at 1, 3, 6, 9, and 12 months after transplantation using flow cytometry, and both UCBT and PBSCT patients were divided into cGVHD and non-cGVHD groups based on whether cGVHD occurred to explore the correlation between the immune cells reconstitution of the two types of transplantation and cGVHD.Results:①The cumulative incidence of the moderate to severe cGVHD in the UCBT group was significantly lower than that in the PBSCT group[9.38% (95% CI 3.35%-15.02%) vs 28.57% (95% CI 9.72%-43.50%) , P=0.008]; the 2-year cumulative incidence of cGVHD and moderate to severe cGVHD in the UCBT group was lower than that in the PBSCT group[15.60% (95% CI 9.20%-23.60%) vs 32.10% (95% CI 15.80%-49.70%) , P=0.047; 10.40% (95% CI 5.30%-17.50%) vs 28.60% (95% CI 13.30%-46.00%) , P=0.014]. ②The absolute counts of CD4 +T cells in the UCBT group were higher than those in the PBSCT group at 6, 9, and 12 months after transplantation[59.00 (36.70-89.65) ×10 7/L vs 31.40 (18.10-44.00) ×10 7/L, P<0.001; 71.30 (49.60-101.45) ×10 7/L vs 41.60 (25.82-56.27) ×10 7/L, P<0.001; 83.00 (50.17-121.55) ×10 7/L vs 44.85 (31.62-62.10) ×10 7/L, P<0.001]; the proportions of CD4 +T cells in the UCBT group were always higher than those in the PBSCT group ( P<0.05) . The absolute counts and proportions of B cells in the PBSCT group were higher than those in the UCBT group at the first month after transplantation[0.70 (0.30-1.70) ×10 7/L vs 0.10 (0-0.30) ×10 7/L, P<0.001; 0.45% (0.30%-2.20%) vs 0.20% (0.10%-0.40%) , P=0.002]; the absolute counts and proportions of B cells in the UCBT group were higher than those in the PBSCT group at 9 and 12 months after transplantation[53.80 (28.00-103.20) ×10 7/L vs 23.35 (5.07-35.00) ×10 7/L, P<0.001; 21.45 (11.80-30.45) % vs 9.00% (3.08%-16.73%) , P<0.001. 66.70 (36.97-98.72) ×10 7/L vs 20.85 (7.72-39.40) ×10 7/L, P<0.001; 22.20% (14.93%-29.68%) vs 8.75% (5.80%-18.93%) , P<0.001]. The absolute counts and proportions of regulatory B (Breg) cells in the UCBT group were higher than those in the PBSCT group at 6, 9, and 12 months after transplantation[1.23 (0.38-3.52) ×10 7/L vs 0.05 (0-0.84) ×10 7/L, P<0.001; 5.35% (1.90%-12.20%) vs 1.45% (0-7.78%) , P=0.002. 2.25 (1.07-6.71) ×10 7/L vs 0.12 (0-0.77) ×10 7/L, P<0.001; 6.25% (2.00%-12.33%) vs 0.80% (0-5.25%) , P<0.001. 3.69 (0.83-8.66) ×10 7/L vs 0.46 (0-0.93) ×10 7/L, P<0.001; 6.15% (1.63%-11.75%) vs 1.40% (0.18%-5.85%) , P<0.001].The absolute counts and proportions of CD3 +T cells, CD8 +T cells, and Treg cells in the UCBT group were not significantly different from those in the PBSCT group. ③The absolute counts of B cells in the non-cGVHD group of UCBT patients were higher than those in the moderate to severe cGVHD group at 6 and 12 months after transplantation ( P=0.038, P=0.043) ; the proportions of B cells in the non-cGVHD group were higher than those in the moderate to severe cGVHD group at 6 months after transplantation ( P=0.049) . The absolute counts of Breg cells in the non-cGVHD group of patients with UCBT were higher than those in the moderate to severe cGVHD group at 6, 9, and 12 months after transplantation ( P=0.006, P=0.028, P=0.050) ; the proportions of Breg cells in the non-cGVHD group were higher than those in the moderate to severe cGVHD group at 9 months after transplantation ( P=0.038) . ④The absolute counts and proportions of B and Breg cells in the non-cGVHD group of patients with PBSCT were not statistically different than those in the moderate to severe cGVHD group. Conclusion:In the process of immune cell reconstitution, the Breg cells in the UCBT group were higher than those in the PBSCT group, and the Breg cells in the non-cGVHD group of the two types of transplantation were always higher than those in the moderate to severe cGVHD group, indicating that Breg cells can reduce the occurrence of cGVHD, revealing the possible reason for the lower incidence of cGVHD in the UCBT group.

Objective:To explore the differences of immune reconstitution between peripheral blood stem cell transplantation and umbilical cord blood transplantation.Methods:A total of 300 patients (aged 18 (8, 33), 163 males and 137 females) with malignant hematological diseases who received allogeneic hematopoietic stem cell transplantation in the First Affiliated Hospital of University of Science and Technology of China from January 2018 to March 2020 were enrolled in this study, including 255 cases of umbilical cord blood transplantation and 45 cases of peripheral blood stem cell transplantation. Multicolor flow cytometry was applied to analyze lymphocyte subsets of the percentages and absolute counts in the two donor types and peripheral blood of patients after receiving hematopoietic stem cell transplantation. The differences between the two grafts were compared, and the lymphocyte subsets results were evaluated at 1, 2, 3, 4, 6, 9, 12, and 18 months after transplantation. 18-month disease-free survival (DFS) within the 300 patients under the two transplantation types were retrospectively analyzed.Results:1. The proportion of NKT cells in peripheral blood group was significantly higher than that in cord blood group (2.79% vs 0.24%, P<0.001). 2. The proportion of helper T cells in the UCBT group was higher than that in the PBSCT group, as well as the counts 6 months after transplantation ( P<0.05). 3. The proportion of NK1 cells (3 rd to 9 th month) and count (4 th to 12 th month) in UCBT group were significantly higher than those in PBSCT group ( P<0.05). 4. NKT cells in the UCBT group were lower than those in the PBSCT group (proportion and count) throughout the monitoring process ( P≤0.001). 5. The proportion of DNT cells (within 1 year) and count (within 6 months) in the UCBT group were significantly lower than those in the PBSCT group ( P<0.05). Conclusions:Compared with the peripheral blood stem cell transplantation group, the umbilical cord blood transplantation patients had a faster rate of lymphocyte reconstitution, and patients received umbilical cord blood transplantation had a stronger ability of immune reconstitution and could achieve long-term hematopoiesis.