Short-term efficacy of venetoclax combined with azacitidine in acute myeloid leukemia: a single-institution experience / 中华血液学杂志

Objective: To explore the safety and short-term efficacy of venetoclax combined with azacitidine (Ven+AZA) in previously untreated patients unfit for standard chemotherapy and patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) in China. Methods: A retrospective study was conducted in 60 previously untreated patients unfit for standard chemotherapy and patients with R/R AML who received Ven+ AZA (venetoclax, 100 mg D1, 200 mg D2, 400 mg D3-28; azacitidine, 75 mg/m(2) D1- 7) at the Peking University Institute of Hematology from June 1, 2019 to May 31, 2021. The incidence of adverse events, complete remission (CR) /CR with incomplete hematological recovery (CRi) rate, objective remission rate (ORR) , and minimal residual disease (MRD) status in patients with different risk stratification and gene subtypes were analyzed. Results: The median age of the patients was 54 (18-77) years, 33 (55.0%) were males, and the median follow-up time was 4.8 (1.4-26.3) months. Among the 60 patients, 24 (40.0%) were previously untreated patients unfit for standard chemotherapy, and 36 (60.0%) were R/R patients. The median mumber cycles of Ven+AZA in the two groups were both 1 (1-5) . According to the prognostic risk stratification of the National Comprehensive Cancer Network, it was divided into 8 cases of favorable-risk, 2 cases of intermediate risk, and 14 cases of poor-risk. In previously untreated patients unfit for standard chemotherapy, after the first cycle of Ven+AZA, 17/24 (70.8%) cases achieved CR/CRi, 3/24 (12.5%) achieved partial remission (PR) , and the ORR was 83.3%. Among them, nine patients received a second cycle chemotherapy and two received a third cycle. Among CR/CRi patients, 8/17 (47.1%) achieved MRD negativity after two cycles of therapy. In the R/R group, after the first cycle of Ven+AZA, 21/36 (58.3%) cases achieved CR/CRi (7/21 achieved MRD negativity) , 3 achieved PR, and the ORR was 66.7%. Among R/R patients, 12 were treated for more than two cycles. There were no new CR/CRi patients after the second treatment cycle, and 14 cases (66.7%) achieved MRD negativity. According to the time from CR to hematological recurrence, the R/R group was divided into 12 cases in the favorable-risk group (CR to hematological recurrence ≥18 months) and 24 in the poor-risk group (CR to hematological recurrence<18 months, no remission after one cycle of therapy, and no remission after two or more cycles of therapy) . Eleven of 24 (45.8%) cases achieved CR/CRi after one cycle of Ven+AZA in the poor-risk R/R group, and 10 of 12 (83.3%) achieved CR/CRi in the favorable-risk R/R group, which was significantly superior to the poor-risk group (P=0.031) . After one cycle of treatment, 13 patients with IDH1/2 mutations and 4 that were TP53-positive all achieved CR/CRi. The CR/CRi rate of 18 patients with NPM1 mutations was 77.8%. Five patients with RUNX1-RUNX1T1 combined with KIT D816 mutation (two initial diagnoses and three recurrences) had no remission. Ven+ AZA was tolerable for AML patients. Conclusion: Ven+AZA has acceptable safety in previously untreated patients unfit for standard chemotherapy, patients with R/R AML can achieve a high response rate, and some patients can achieve MRD negativity. It is also effective in NPM1-, IDH1/IDH2-, and TP53-positive patients. The long-term efficacy remains to be observed.

Chinese Medicine Might Be A Promising Way for A Solution to Arsenic Nephrotoxicity / 中国结合医学杂志

Arsenic from environmental contamination is a risk factor for kidney disease, and the clinical use of arsenic also triggers a new concern that the potential kidney damage caused by exposure to clinical doses of arsenic trivalent. So far, the undergoing mechanisms contributing to arsenic nephrotoxicity mostly remain unclear, and universally accepted methods for preventing this complication are limited too. Ancient Chinese medical scientists recognized the toxicity of arsenic long ago, and there were some records of Chinese herbs against arsenic poisoning in ancient books of Chinese medicine. In the past decade, several herbal formulations, as well as some potentially active compounds extracted from Chinese herbs, have been employed to prevent arsenic nephrotoxicity both in vivo and in vitro and showed better therapeutic effects. The present paper thus summarizes and discusses these Chinese medicine methods in preventing such a public health problem. In addition, we call for large, well-designed, randomized, and controlled clinical trials to be performed to further assess the efficacy and safety of these potential methods of Chinese medicine against arsenic nephrotoxicity.

Application of intestinal CT texture analysis and nonlinear discriminant analysis in differential diagnosis of colorectal cancer and ulcerative colitis / 上海交通大学学报（医学版）

Objective·To evaluate the value of texture analysis in the discrimination of colorectal cancer (CRC) and ulcerative colitis (UC). Methods?·?The CT images of 61 CRC patients, 62 UC patients and 42 control objects were retrospectively analyzed. All the patients were pathologically proved and performed triphasic contrast-enhanced CT scan: non-enhanced phase (NP), the arterial phase (AP) and the enteric phase (EP). The region of interest was drawn along the abnormal bowel wall’s edge in each scan phase and texture features were generated by MaZda software. Based on 3 texture feature selection methods, the optimal subsets were generated and analyzed by 6 texture feature classification methods. The results were shown by misclassification rate (MCR). To compare the performance of texture-based classification and human visual classification, two radiologists with more than 10 years of gastrointestinal disease diagnostic experience analyzed the data. Results?·?The texture analysis based average MCR of differentiation between CRC and UC was (28.42±6.89)%, (28.19±4.07)%, (19.10±3.58)% in NP, AP, EP respectively. Compared with other texture feature classification methods, nonlinear discriminant analysis (NDA) was more accurate. In EP, NDA achieved an excellent classification result (MCR=12.61%). The average MCR between CRC and normally dilated bowel wall (NOR) was (13.33±7.21)%, (15.49±5.47)%, (6.74±3.02)%, while the average MCR between UC and NOR was (19.26±4.68)%, (20.04±6.63)%, (16.74±6.36)% in NP, AP and EP respectively. For visual classification between CRC and UC, the average MCR was (40.48±3.21)%, (35.71±1.60)%, (26.43±1.15)% in NP, AP, EP respectively. But the MCR of texture classification was lower than that of human vision classification, and computer texture classification had higher differential diagnosis rate. Conclusion?·?The CT-based texture analysis could be a feasible supplementary method to differentiate CRC from UC. NDA is more accurate than other classification methods, especially in EP.

Changes of blood glucose and glycosylated hemoglobin in patients with progress of ischemic stroke / 中国综合临床

Objective To observe the changes of fasting plasma glucose (FPG) and glycated hemoglobin(HbAcl) in patients with progressive ischemic stroke(SIP),and to provide scientific basis for the prevention and treatment of SIP.Methods Two hundred and twenty-one cases of acute ischemic stroke in Aerospaceplane General Hospital of Beijing were enrolled in this study.They were divided into progressive ischemic stroke group(123 cases) and non-progressive ischemic stroke group(98 cases) according to the NIHSS score.The changes of FPG and FPG were observed and compared between the two groups after admission to the hospital.Results The incidence of diabetes in group SIP (51.22％,63/123) was significantly higher than that in non SIP group(15.31％,15/98),and the difference was statistically significant(P=0.000).The FPG and HbAlc of SIP group were (8.35± 3.76) mmol/L,(7.31 ± 2.07) ％,of non SIP group were (6.47± 2.86)mmol/L,(6.25± 1.31)％,the difference between the two groups was statistically significant (P =0.000).Distribution rate of FPG＜6.1 mmoL/L,6.1 mmol/L ≤FPG＜7.0 mmol / L,FPG≥7.0 mmol/L in SIP group patients were 38.21％,14.64％,47.15％ respectively,in non SIP group were 64.39％ and 16.33％,19.39％ respectively,the differences between the two groups were statistical significance(P=0.000).Distribution rate of HbA1c≤7.0％,7.0％＜HbA1c ≤.0％,HbA1c＞9.0％ of patients in the SIP group were 53.66％ (66/123),25.20％(31/123),21.14％(26/123) respectively,and in non SIP group were 79.59％(78/98),16.33％(16/98),4.08％ (4/98) respectively,the differences between the two groups were statistical significance (P =0.000).Conclusion The occurrence and development of SIP is not only related to diabetes,but also closely related to blood glucose and HbA1c levels.High blood glucose and high levels of HbA1c have important significance for early identification and prediction of SIP.

Effect of Chuanhuang No. 1 recipe on renal function and micro-inflammation in phase 3 chronic kidney disease patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of Chuanhuang No.1 Recipe (CHR) on renal function and micro-inflammation in phase 3 chronic kidney disease (CKD) patients.</p><p><b>METHODS</b>Totally 60 phase 3 CKD patients were randomly assigned to the treatment group (treated by CHR) and the control group (treated by Losartan Potassium), 30 in each group. All patients received basic treatment. Patients in the treatment group took CHR decoction, 400 mL each time, one dose per day, while those in the control group took Losartan Potassium, 50-100 mg per day. All medication lasted for 24 weeks. Changes of serum creatinine (SCr), blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), serum uric acid (UA), 24 h urinary protein excretion (24 h U-pro), urinary microalbumin (U-Alb), high-sensitivity C-reactive protein (hs-CRP), serum tumor necrosis factor (TNF)-alpha, and serum IL-6 were detected and compared before and after treatment. Efficacy was also compared.</p><p><b>RESULTS</b>Compared with before treatment, SCr and BUN significantly decreased in the treatment group (P<0.05, P<0.01); eGFR in- creased (P<0.05). Only UA obviously decreased in the control group (P<0.05), but with no obvious change in SCr, BUN, or eGFR. Compared with before treatment, 24 h U-pro decreased after treatment in the treatment group (P<0.05), but with less decreased level when compared with the control group. U- Alb was also significantly decreased in the control group (P<0.01). There was statistical difference in 24 h U-pro and U-Alb between the two groups after treatment (P<0.05). Compared with before treatment, hs-CRP obviously decreased after treatment in the two groups, but serum levels of TNF-alpha and IL-6 obviously decreased only in the treatment group (P<0.05). The total effective rate was obviously higher in the treatment group than in the control group (70.00% vs. 43.33%, P<0.01).</p><p><b>CONCLUSION</b>CHR could efficiently improve the renal function of phase 3 CKD patients and alleviate the micro-inflammation.</p>

Biological characteristics of wharton's jelly derived mesenchymal stem cells after cryopreservation / 中国实验血液学杂志

Aim of this study was to explore the effects of cryopreservation on biological characteristics of wharton's jelly derived mesenchymal stem cells (WJ-MSC), and to provide experimental evidence for clinical applications and the establishment of WJ-MSC bank. Primary WJ-MSC were produced by umbilical cord tissue culture in vitro. Fifth passage of WJ-MSC acquired by continuous cell culture were mixed with cryoprotectants, frozen in -80°C refrigerator and stored in liquid nitrogen. After the cryopreserved WJ-MSC were thawed, the first passage of WJ-MSC was obtained through cell culture and was taken as the 1st preserved passage (PP1). Thus, PP2-PP15 WJ-MSC were obtained by continuous cell subculture. The 1st control passage (CP1) to 15th passage (CP15) represented the 6th passage to 21st passage WJ-MSC acquired by subculturing in non-cryopreserved group. The biological characteristics of WJ-MSC from cryopreserved and control group, including the recovery rate of nucleated cells, trypan blue exclusion, CCK-8 activity, cell apoptosis, cell adherence, proliferation index, cell surface antigen, cell cycle and the capacities of induced differentiation into adipocyte, osteoblast and neuron, were detected and compared. The results indicated that the recovery rate of nucleated cells of cryopreserved WJ-MSC was 98.2%, trypan blue exclusion rate was 94.3%, CCK-8 activity was 91.4%, apoptotic rate was 3.9%, and the adherence rate was 92.6%. There was a statistically significant difference in proliferation index between PP1 and CP1 (P < 0.05), but there were no statistically significant differences between PP2-PP15 and their corresponding controls. The subculture cells highly expressed CD29, CD44, CD71, CD73, CD90, CD105, CD166 and HLA-ABC, and lowly expressed CD34, CD45 and HLA-DR. The expressions of above-mentioned surface antigens were not different statistically between two groups. The proliferation latency and logarithm proliferation of the subculture cells between two groups were also not different. After induced differentiation into adipocyte, osteoblast and neuron, the staining with oil red O, alkaline phosphatase and neuron-specific enolase was performed respectively, and the positive degrees were not clearly different macroscopically between two groups. Relatively high levels of triglyceride, alkaline phosphatase, and neuron-specific enolase in relevant cells could be detected, but had no significant differences between two groups. It is concluded that some WJ-MSC (< 10%) are damaged after cryopreservation, and the biological characteristics of WJ-MSC in cryopreservation group keep constant, as compared with that in non-cryopreservation group.

Dynamic changes in PD-1, TLR3, and TLR4 surface expression on peripheral blood mononuclear cells in chronic hepatitis C patients undergoing peg-IFNalpha-2a plus ribavirin combination therapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic changes in expression of programmed death (PD)-1, Toll-like receptor (TLR)3, and TLR4 on the surface of peripheral blood mononuclear cells (PBMCs) in patients with chronic hepatitis C (CHC) that occur in response to pegylated-interferon alpha-2a (peg-IFNalpha-2a) plus ribavirin (RBV) combination therapy, and to analyze the relation to achievement of sustained virological response (SVR). METHODS Twenty-three CHC patients and 10 healthy controls were enrolled in the study. All CHC patients underwent 48 weeks of combination therapy with peg-IFNalpha-2a (180 microg, subcutaneous injection, once weekly) plus RBV (15 microg/kg, oral, once daily). Total PBMCs were isolated from both groups (CHC patients at treatment week 0, 12, 24, and 48 and post-treatment week 24; controls at enrollment) and subjected to flow cytometric analysis of PD-1, TLR3, and TLR4 surface expression. In addition, serum levels of alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels were analyzed by enzymatic assay and the AmpliPrep/COBAS (Roche) nucleic acid amplification test, respectively. SVR was defined as undetectable levels of HCV RNA at post-treatment week 24. Intergroup differences were assessed by one-way ANOVA.</p><p><b>RESULTS</b>The expression ratios of PD-1, TLR4 and PD-1: TLR4 on PBMCs were significantly higher in CHC patients before therapy than in the healthy controls (45.20 +/- 7.12% vs. 16.82 +/- 4.13%, 58.45 +/- 15.13% vs. 21.09 +/- 2.89%, and 35.54 +/- 7.69% vs. 14.12 +/- 2.89%; all P < 0.05). In contrast, the expression ratios of TLR3 and PD-1:TLR3 were slightly, but not significantly, higher in CHC patients before therapy than in the healthy controls (P > 0.05). During the course of peg-IFNalpha-2a plus RBV combination therapy, the expression ratios of PD-1 and TLR4 on PBMCs showed a decreasing trend, while TLR3 expression showed an increasing trend. Furthermore, CHB patients who achieved SVR at post-treatment week 24 had a significantly different expression ratio of PD-1 and TLR3 than those who did not achieve SVR (P < 0.05).</p><p><b>CONCLUSION</b>Surface expression of PD-1, TLR4, and PD-1:TLR4 is up-regulated in the total PBMCs of CHC patients. Peg-IFNalpha-2a plus RBV treatment-induced suppression of HCV replication results in a significant reduction in PD-1 and TLR4 expression on the surface of PBMCs, but a remarkably elevated level of TLR3 expression. The dynamic change in PD-1 and TLR3 expression on PBMCs that occurs during antiviral therapy may be related to achievement of SVR.</p>

Hepatitis C virus strain JFH1 down-regulates expression of growth arrest and DNA damage-inducible gene 45a in human hepatoma Huh7.5.1 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of hepatitis C virus (HCV) strain JFH1 on expression of the human gene, growth arrest and DNA damage-inducible gene 45 alpha (GADD45a), in infected hepatoma cells.</p><p><b>METHODS</b>HCV JFH1 RNA-containing supernatants were used to infect the human hepatoma cell line, Huh7.5.1; infection was confirmed by Western blot detection of the HCV-encoded non-structural 5A (NS5A) protein and core protein. Infection-induced changes in GADD45a mRNA and protein expressions were measured by real time PCR using SYBR Green and Western blotting, respectively. Significance of differences between the levels detected in JFH1-infected or uninfected Huh7.5.1 cells was analyzed by single factor analysis of variance testing.</p><p><b>RESULTS</b>The HCV infection system was successfully established, as evidenced by expression of NS5A protein and core protein. The GADD45a mRNA and protein levels were significantly down-regulated in JFH1-infected Huh7.5.1 cells, by 0.57+/-0.09 and 0.28+/-0.03, respectively, as compared to levels in uninfected Huh7.5.1 cells (F values were 75.407 and 560.04, respectively; P less than 0.01).</p><p><b>CONCLUSION</b>HCV inhibits the mRNA transcription and protein expression of host GADD45a, which may contribute to the pathogenesis of hepatocellular carcinoma caused by HCV infection.</p>

Morphologic diagnosis and onset characteristics of acute leukemia: a retrospective analysis of 233 cases in 10 years / 中国实验血液学杂志

The objective of this study was to evaluate the value of morphologic diagnosis for acute leukemia (AL), to explore the relation of morphologic diagnosis with immunology, cytogenetics and molecular biology diagnosis of AL and to analyze the onset characteristics of AL in 10 years. The samples of bone marrow and peripheral blood from 233 newly diagnosed cases of AL were collected during 2001-2011 years; the morphologic examination and immunologic, cytogenetic and molecular biologic examination (ICM) were carried out, the consistency of morphologic diagnosis with ICM diagnosis was compared, the onset characteristics of AL was analyzed. The results showed that: (1) the consistent rate of immunology, cytogenetics, molecular biology diagnosis with morphologic diagnosis was 84.3%. The order of consistent rat was AUL, M0 < M1 < HAL < M4 < M2 < M3 < M5 < ALL < M6, M7, AP; (2) Misdiagnosis always occurred among AUL, M0, M1, ALL and HAL or among M2a, M3v, M4 and M5. (3) In 233 cases, the highest ratio of blast was observed in M1 (92.5%), while the lowest ratio of blast was observed in M2 (49.5%). (4) AL occurred more frequently in males than that in female (147:86). (5) AL occurred in patients aged from 1 to 88 years. The median age was 41.5 for AUL, 40.8 for M0, 43.4 for M1, 46.3 for M2, 33.8 for M3, 42.6 for M4, 48.8 for M5, 77.3 for M6, 2.5 for M7, 65.0 for AP, 29.1 for ALL and 40.3 for HAL. (6) The number of patients in the later five years (139 cases) was significantly greater than that in the first five years (94 cases), especially the patients with M1, M2, M3, M4, and M5. It is concluded that morphologic diagnosis has important clinical value in the MICM diagnosis of AL. Attaching importance to the confusing cell morphology and onset characteristics of AL can improve the diagnostic accuracy.

The therapeutic effect of Anluohuaxian capsule combined with adefovir dipivoxil on patients with chronic hepatitis B and influence on hepatic histology / 中华肝脏病杂志

<p><b>UNLABELLED</b>To observe the efficacy of adefovir dipivoxil(ADV) in combination with Anluohuaxian capsule in the treatment of chronic hepatitis B (CHB) patients.</p><p><b>METHODS</b>72 cases with CHB were randomly divided into two groups. 36 cases of treatment group were given ADV combined with Anluohuaxian capsule for 48 weeks. 36 cases of control group were given ADV. The levels of serum ALT, AST, Alb, TBil, HA, LN, CIV, HBV DNA and hepatic tissue were compared before and after being treated.</p><p><b>RESULTS</b>After 48 weeks treatment,the liver function, serum fibrosis index and histology of treatment group and control group all have improved. After treatment, the two groups in the levels of ALT(t=0.746, P=0.342), AST (t=0.369, P=0.713), TBil (t=0.146, P=0.684), Alb(t=0.148, P=0.883), liver tissue inflammation mobility scoring (t=1.666, P=0.100) and HBV DNA negative rate (x2=0.141, P=0.708) were no evident difference.The level of HA, LN, CIV were significantly lower in treatment group(101.58+/-30.11, 147.89+/-41.72, 38.75+/-9.50) compared with control group(182.25+/-117.59, 181.50+/-56.96, 74.92+/-31.14) (P less than 0.05). After the treatment, the liver tissue fibrosis scoring was significantly lower in treatment group (10.61+/-2.37) compared with before the treatment (12.28+/-3.16) (P less than 0.05).There was no difference found between after the treatment (11.36+/-2.93) and before the treatment (12.17+/-3.01) in control group (P more than 0.05).</p><p><b>CONCLUSIONS</b>The results show that the treatment with ADV in combination with Anluohuaxian capsule can play promoting antifibrotic effect and significant improved liver histology of chronic hepatitis B patients.</p>

The effects of thrombopoietin on the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes / 中国应用生理学杂志

<p><b>OBJECTIVE</b>In order to investigate whether or not thrombopoietin (TPO) could promote the fibrogenesis of bone marrow stromal cells in absence of megakaryocytes (MKs).</p><p><b>METHODS</b>Improved dexter culture system with various TPO concentrations was used for ex vivo culture of bone marrow stromal cells. Relative proliferation index, the expressions of fibronectin, laminin and type IV collagen, and the systhesis of type III procollagen were detected at different time points during culture process.</p><p><b>RESULTS</b>TPO stimulated the proliferation of bone marrow stromal cells. Relative proliferation index of the stromal cells increased with the TPO concentration increasing, and was not related to the exposure time. The expressions of fibronectin, laminin, and type IV collagen appeared stronger in the TPO groups than those in the control group. But the expressions of these molecules were not dependent upon the culture time. TPO could accelerate the synthesis of type III procollagen in bone marrow stromal cells, and this acceleration was unrelated to the TPO concentration.</p><p><b>CONCLUSION</b>These findings suggested that TPO could stimulate the stromal cells with a consequence of increased syntheses and secretions of the extracellular matrix and collagen in absence of MKs. In other words, TPO could promote the fibrogenesis of bone marrow stromal cells without the existence of MKs.</p>

HCV NS5A protein down-regulates hepcidin gene expression and increases hepatic intracellular iron storage / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.</p><p><b>METHODS</b>HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.</p><p><b>RESULTS</b>HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.</p><p><b>CONCLUSIONS</b>HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.</p>

In vitro activity of human bone marrow cells after cryopreservation in liquid nitrogen for 21 - 25 years / 中国实验血液学杂志

The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the frozen methods on the cryopreservative effectiveness was little. It is concluded that the human bone marrow cells with DMSO-AuP or DMSO-HES-HuA as cryoprotectant, frozen by a programmed freezer or -80 degrees C refrigerator, could be then preserved in liquid nitrogen for long time. When the preserving time was as long as 21 to 25 years, the morphology, the recovery rate and the activity of various kinds of cells were still good. The method of freezing by -80 degrees C refrigerator with 5% DMSO-6% HES-4% HuA and preserving in liquid nitrogen would be convenient, cheap and easily-manipulated for preservation of the human bone marrow cells.

Cationic liposome-mediated transfection of CD40 ligand gene inhibits hepatic tumor growth of hepatocellular carcinoma in mice / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To evaluate the efficacy of cationic liposome-mediated CD40 ligand (CD40L) gene therapy for hepatocellular carcinoma.</p><p><b>METHODS</b>1x10(6) of parental H22 cells or H22 cells transfected with the expression vector containing murine CD40L cDNA encoding the entire coding region (pcDNA3.1(+)-mCD40L) were inoculated subcutaneously into the left flanks of syngenic BALB/C mice. The tumor-bearing mice (tumor nodules 10 mm in maximal diameter) received the treatment of the intratumoral injection of pcDNA3.1(+)-mCD40L/Transfectam, pcDNA3.1(+), or phosphate-buffered saline (PBS), or no treatment. The mice were monitored for tumor growth weekly. We examined mCD40L messenger ribonucleic acid (mRNA) expression by reverse transcription polymerase chain reaction (RT-PCR) and the histologic changes in tumors at two weeks after intratumoral injection using immunohistochemical staining of tumor tissues.</p><p><b>RESULTS</b>All mice inoculated with parental H22 cells developed a tumor subcutaneously, and the tumor size increased progressively within three weeks. However, the mice receiving H22-CD40L cells exhibited complete regression of the tumor two weeks after tumor cell inoculation. The tumor-bearing animals with the treatment of pcDNA3.1(+) or PBS, or without treatment had progressive tumor growth, while those mice treated with pcDNA3.1(+)-mCD40L exhibited a significant inhibition of tumor growth. RT-PCR analysis showed that 783-bp fragments corresponding to the mCD40L mRNA were amplified only from pcDNA3.1(+)-mCD40L treated tumors. The tumor samples from pcDNA3.1(+)-mCD40L-treated mice showed significant lymphocyte infiltration, apoptotic bodies, and confluent necrosis in the tumor tissues.</p><p><b>CONCLUSION</b>The tumorigenicity of CD40L-expressing cells was abrogated when the cells were implanted subcutaneously. In vivo gene therapy of established liver tumor nodules in mice by the intratumoral injection of pcDNA3.1(+)-mCD40L led to significant tumor inhibition. There was mCD40L mRNA expression in the tissues from pcDNA3.1(+)-mCD40L-treated tumors. The intratumoral injection of pcDNA3.1(+)-mCD40L induced a strong inflammatory, mainly lymphocytic infiltration of the tumor, and increased the necrotic rate of the neoplastic cells.</p>

Influences of bone marrow mesenchymal stem cells in patients with acute myeloid leukemia and non-leukemia on HL-60 cells -- a comparison study / 中国实验血液学杂志

This study was aimed to compare the influences of bone marrow mesenchymal stem cells (BMMSCs) from patients with acute myeloid leukemia (AML), AML patients with complete remission (CR) and non-leukemia patients on HL-60 cells. The HL-60 cells were divided into three groups: group of co-cultivation with BMMSCs of AML patients, group of co-cultivation with BMMSCs of AML patients with CR and group of co-cultivation with BMMSCs of non-leukemia patients. The count of HL-60 cells, the CD11b and survivin expression of HL-60 cells, the cell cycle distribution of the HL-60 cells in 3 groups were compared by flow cytometry, the morphology and differentiation rate of HL-60 cells in 3 groups were observed and compared by microscopy. The results showed that there were no differences in HL-60 cell count at five and seven days, in HL-60 distribution at the G(0)/G(1) phase, in survivin and CD 11b expressions in 3 groups. All cells of 3 groups began to mature, and the differentiation rates in 3 groups were 18.0 +/- 3, 17.0 +/- 1.3 and 19.0 +/- 2.0 respectively, therefore there were no significant differences between the 3 groups (p = 0.23). It is concluded that there is no influence of BMMSCs in 3 groups on the proliferation and differentiation of HL-60 cells.

Comparison of biological characteristics of bone marrow mesenchymal stem cells in acute myeloid leukemia with those from non-leukemia / 中国实验血液学杂志

This study was aimed to compare partial biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) in AML patients, AML patients with complete remission (CR) and non-leukemia patients. The bone marrow (BM) MSCs were divided into 3 groups: group of MSCs from AML patients, group of MSCs from AML patients with CR, group of MSCs from non-leukemia patients. The morphologic features of MSCs were observed by light microscopy; CFU-F numbers of MSCs were counted after Wright-Giemsa staining in situ; the fusion times of MSCs were determined; the growth curves of MSCs were drawn by counting cell numbers; the immunophenotypes and cell cycle of MSCs were detected by flow cytometry; the DI values of MSCs were calculated. The results showed that the morphologic features of MSCs in 3 groups did not display difference; there was significant difference (p < 0.01) of CFU-F numbers in 3 groups, while CFU-F number of MSCs in AML group was minimal; there was significant difference of MSC fusion time in 3 groups, while fusion time of MSCs in AML group was most long; the growth curves of MSCs in 3 groups were similar; MSCs in 3 groups highly expressed CD105 and CD106, but not expressed CD45; the cell distribution ratios at phase of G(0)/G(1) for MSCs in 3 groups were 89.9 +/- 4.0%, 90.2 +/- 3.0% and 91.0 +/- 3.0% respectively; the DI values of MSCs in 3 groups were between 0.9 and 1.1. It is concluded that no significant difference of biological characteristics of the second generations of MSCs is found between those in leukemia and non-leukemia patients.

Comparison of different cryopreservation systems for peripheral blood stem cells / 中国应用生理学杂志

<p><b>AIM</b>To explore proper cryopreservative systems for hematopoietic stem cells.</p><p><b>METHODS</b>Peripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.</p><p><b>RESULTS</b>The recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.</p><p><b>CONCLUSION</b>5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.</p>

Effects of recombinant human thrombopoietin on stromal cells in culture in vitro / 中国实验血液学杂志

This study was aimed to investigate whether the thrombopoietin (rhTPO) may facilitate myelofibrosis or not. The modified Dexter culture system with various concentrations of rhTPO was used to culture the stromal cells in vitro; the proliferative activity of cells was detected by MTT method; the morphologic changes were observed by light and scanning electron microscopy; the staining changes of ALP, PAS, AS-D NCE and IV type collagen were observed by cytochemistry method; the changes of fibronectin, laminin and IV type collagen were assayed by immunohistochemistry method; the cell surface antigens were assayed by flow cytometry. The results indicated that rhTPO could promote the proliferation of stromal cells which was related to the concentrations of rhTPO. Proliferative activity of stromal cells increased with increasing of rhTPO concentration, and was not related to the exposure time. On day 3 stromal cells adhered to the wall, and became oval. On day 7 stromal cells turned to fusiform and scattered dispersively. On day 12 to 14 these cells ranged cyclically and became long fusiform. Cells covered 70%-80% area of bottle bottom at that time. By day 16 to 18 these cells covered more than 90% area of bottom and ranged cyclically. They displayed the same shape as fibroblasts. By light microscopy with Wrights-Giemsa staining, fibroblasts predominated morphologically, few macrophages, endothelial cells and adipose cells were found. There were no significant differences between experimental group and control group. On day 14 to 42 the adherent cells were positive with PAS staining, poorly positive with ALP and naphthol AS-D chloroacetate esterase (AS-D NCE) staining, and the difference in cytochemistry was not significant between two groups. When these cells were dyed with Masson's trichrome and Gomori's staining, neither collagen fibers nor reticular fibers were positive, but fibronectin, laminin, and collagen type IV appeared positive stronger in experimental group than those in control. The expressions of these molecules were not dependent on culture time. By scanning electron microscopy microvilli and fibers on cell surface appeared more and more, monolayer cells evolved into multilayer cells, and newly-formed fibroblasts appeared gradually as culture time prolonged. These alterations were not different among various groups. The expressions of CD34, CD45, CD105, CD106, and CD166 were not affected obviously by rhTPO. It is concluded that rhTPO had no effects on histochemical properties of stromal cells. Fiber staining and scanning electron microscopic examinations revealed that rhTPO can not facilitate fiber formation of stromal cells. But rhTPO may be able to augment the expressions of fibronectin, laminin and collagen type IV of stromal cells. Therefore it is still necessary to follow up the patients for a long time, who have received rhTPO therapy clinically.

Influence of cryopreservation on leukemic dendritic cells derived from leukemia patients / 中国实验血液学杂志

This study was aimed to investigate the influence of cryopreservation on biological properties and function of leukemic dendritic cells (L-DCs) derived from patients with acute or chronic leukemia. Some fresh leukemic cells were detected immediately; some were cultured immediately; some were cryopreserved in -80 degrees C with 5% DMSO-6% HES as cryopreservor. After being thawed, they were cultured. The combination of rhGM-CSF, rhIL-4, rhTNF-alpha and other cytokines were added into the culture system. 12 days later, L-DCs were assayed for morphology, immunophenotype, mixed lymphocytic reaction (MLR) and CTL cytotoxicity on autologous leukemic cells. The results showed that both fresh and cryopreserved leukemic cells obtained from patients with acute or chronic leukemia revealed typical DC morphologically by means of using combinations of cytokines in culture, but there was no significant difference between pre-or post cryopreservations. L-DCs also upregulated the expression of CD80, CD54, HLA-DR, CD1a, CD83 and CD86, and downregulated the expression of CD14, but there was also no difference as compared with L-DCs befor cryopreservation. L-DCs derived from leukemic cells were also capable of stimulating MLR and inducing CTL which could kill autologous leukemic cells obviously. It is concluded that leukemic cells, regardless of fresh or frozen, can induce L-DCs after culture with cytokine combination. The L-DCs can induce CTL targeting autologous leukemic cells, and may be used to treat MRD as immunotherapy. The induction and biological properties of L-DCs are not influenced by cryopreservation.

Neurotoxicity Mechanisms of Thrombin in Intracerebral Hemorrhage / 国际脑血管病杂志

The thrombin is a neurotoxic agent,which plays an important role in the course of brain edema and brain injury following intracerebral hemorrhage.This article reviews the neurotoxicity mechanisms of thrombin in intracerebral hemorrhage.