Objective To explore the influence of cryopreservation by ladder-style freezing from low temperature refrigerator to liquid nitrogen on hemopoietic stem cell(HSC)transplantation.Methods From April 2001 to April 2006,31 cases treated with HSC transplantation were randomized to controlled-rate freezer-liquid nitrogen group(n=15)and refrigerator-liquid nitrogen group(n=16)in Department of Hematology of 1st People's Hospital of Yunnan Province.The hemopoietic reconstitution time was observed,and the viability of HSC was determined by trypan blue exclusion test in two groups.Results No significant differences were found in transfusion values of mononuclear cells(MNC)and CD34+ cells,rates of trypan blue exclusion and time of ANC0.05).Conclusion Cryopreservation of HSC can produce successful engraftment by ladder-style freezing which has cryoprotectant solution containing final concentrations of 3-percent hydroxyethyl starch,4-percent human serum albumin,and 5-percent DMSO,and its effect is the same as traditional controlled-rate method with 10-percent DMSO cryoprotectant.The new freezing procedure is faster and easier,and freezing cost is reduced.

Objective To investigate the effects of wortmannin (WT) on leukemia cells cycle and BCL-2 protein expression. Methods 0 25?mol/L PI-3 kinase inhibitor wortmanin was used to treat K562 cells for 24 hours, and Arac was simultaneously used to induce the cell apoptosis. The specific fluoresent staining and FCM analysis were adopted to measure the cell cycle distribution and BCL-2 expression. Results Compared with the control cells, the proliferation index(PI) of the K562 cells treated by wortmannin was significantly lower, the cell number of G1 phase and the percentage of cell apoptosis increased, and the BCL-2 protein expression significantly decreased. Conclusion Wortmannin could inhibitedtheproliferationofK5 6 2cells ,andwascellcyclespecificagent (CCSA) .

BACKGROUND: Uncontrolled-rate freezing in -80 ℃ refrigerator is convenient, while controlled-rate freezing in -196 ℃ liquid nitrogen is reliable and long-term, the combination of the two can simplify the process and has been successfully used in clinics. OBJECTIVE: To explore the influences of different cryoprotectants by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen on the cryopreservation of hemopoietic stem cells. METHODS: The experiments were divided into four groups: 10% dimethyl sulfoxide (DMSO) group, 5% DMSO combined with 3% hydroxyethyl starch group, 5% DMSO combined with 0.25 mol/L trehalose group, 5% DMSO combined with 3% hydroxyethyl starch and 0.25 mol/L trehalose group. Peripheral hemopoietic stem cells were cryopreserved by ladder-style freezing from -80 ℃ low temperature refrigerator to liquid nitrogen. The ultrastructural changes were examined by transmission electron microscopy, the expressions of Annexin-V, PI and Caspase-3 were detected by flow cytometry. RESULTS AND CONCLUSION: There was no significant difference in survival rate, apoptotic rate and necrotic rates of the cryopreserved cells in the four groups (P > 0.05). The ultrastructural changes had no significant difference under the transmission electron microscopy. The viability was more than 90% in frozen-thawed mononuclear cell colonies, and the apoptosis was roughly 50% in the frozen-thawed CD45+ cell population, which contained many mature cells. Of hemopoietic stem cells, early stage cells have greater resistance to damage of cryopreservation than late stage cells. It is concluded that the addition of hydroxyethyl starch or trehalose into DMSO exhibits no synergistic protective effect on the cryopreservation of hemopoietic stem cells.

AIM:To explore the preliminary mechanism of senegenin ( Sen) on inhibiting hypoxia/reoxygenation ( H/R)-induced apoptosis of primary cortical neurons .METHODS:The cultured cortical neurons were randomly divided in-to normal group (control group), model group (H/R group), Sen+H/R group and Sen group.Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis .The protein levels of JNK , p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting .RESULTS:The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen +H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully .The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05).CONCLUSION:Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax .

Objective To investigate the role of cerebrospinal fluie( CSF)cytology on eiagnosis of meningeal cancer. Methods Retrospectively analyzee the clinical eata of 23 cases with meningeal cancer. Results (1)Of 23 patients,CSF eetection of 17 cases were showee with cancer cells at the first lumbar puncture,3 cases at the 2ne lumbar puncture,2 cases at the 3re eetection,ane 1 case at 4th eetection.(2)Of 23 cases with cerebrospinal fluie cytology positive patients,17 cases were carriee cranial MRI scan. The MRI showee that 9 were normal ane 8 cases were brain parenchyma ane meningeal image enhance. Ten cases were performee the enhancee MRI scan,5 cases were with extensive meningeal thickening,3 cases were showee meningeal extensive enhancement of brain surfer ane intracranial cerebral sulci,1 case was the circular shaeow strengthen at eifferent size at next to the eouble lateral parietal,occipital ane left cerebella hemisphere,ans 1 case was with tough brain surface ane abnormally long T1,long T2 signal at lateral ventricles,thire ventricle, fourth ventricle epeneymal.(3)The relationship between the primary tumor ane cancer eetection in cerebrospinal fluie:19 patients were foune with primary tumor lesions,inclueing 5 cases leukemia(3 cases with acute lymphoblastic leukemia,2 cases with acute non-lymphocytic leukemia),4 cases with lung cancer,3 cases with breast cancer,2 cases with brain lymphoma,1 case with melanoma,1 case with Hoegkin′s lymphoma,l case with nasopharyngeal carcinoma, 1 case with vaginal squamous cell carcinoma, 1 case with gastric carcinoma. Conclusion Multiple cerebrospinal fluie cytology eetection may improve meningeal cancer eetection rate. CSF cytology eetection can improve eiagnosis rate of meningeal cancer. No relationship between the eetection of cancer cells in cerebrospinal fluie ane brain MRI meningeal lesions,ane the further research neee to be eone with expaneing the sample size.

AIM: To prepare glycine liposome microparticle and observe the effect of glycine liposomes on cardiomyocyte injury induced by hypoxia/reoxygenation. METHODS: (1) Reverse-phase evaporation method was used to produce glycine liposomes, the effects of different organic solvents: aether, chloroform and two mixtures of aether/chloroform on entrapment efficiency were evaluated, transmission electron microscope was used to detect the particle diameter of glycine liposomes. (2) A cardiomyocyte injury model was established by using hypoxia/reoxygenation, the activities of lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) of each group were detected. RESULTS: The entrapment efficiency of glycine liposomes prepared with the mixtures of aether/chloroform is highest compared with other organic solvents (64.8%, P

AIM: To study the expression of glycine receptor (GlyR) in adult rat cardiac tissue and cultured cardiomyocytes from neonatal rat hearts. METHODS: Total RNA and membrane protein were extracted from cardiac tissue of adult rats and primary cultured cardiomyocytes from neonatal rat hearts, ?1 and ? subunits mRNA and protein of GlyR were detected with the method of reverse transcription nest DNA polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: The mRNA and protein expression of ? subunit were identified in the adult rat heart tissue and cultured cardiomyocytes, similar to the sequence and immunogenicity generated from GlyR ? subunit of the rat spinal cord; for ?1 subunit, mRNA was found in only cultured cardiomyocytes. CONCLUSION: These data demonstrate that adult rat cardiac tissue and cultured cardiomyocytes from neonatal rat hearts express mRNA and protein of GlyR subunits similar to the GlyR that expresses in the spinal cord, suggesting that GlyR exists in the membrane of the rat cardiomyocytes.

AIM: To investigate whether glycine receptor is involved in the protection of glycine against(anoxia/reoxygenation) injury in cardiomyocytes by detecting oxygen free radical metabolism,apoptosis and intracellular calcium overload.METHODS: The neonatal rat cardiomyocytes were cultured and exposed to anoxia and reoxygenation((A/R)) in the presence of glycine receptor antagonist,glycine or in free chloride buffer.The superoxide dismutase(SOD) activity,the contents of malondialdehyde(MDA) and nitric oxide(NO),the intracellular free calcium concentration and the apoptotic rate in the cardiomyocytes were determined.RESULTS: SOD activity and NO content in cardiomyocytes were lower,but MDA content,intracellular free calcium concentration and apoptotic rate in cardiomyocytes were higher in A/R group than those in control.Pretreatment with glycine inhibited the above changes caused by A/R,which was reversed by strychnine treatment and in the free chloride medium.CONCLUSIONS: Glycine inhibits free radical production,attenuates calcium overload,decreases apoptotic rate and increases SOD activity and NO release in cardiomyocytes exposed to(A/R).These findings suggest that glycine exerts a protective effect against A/R injury via glycine receptor and glycine protects the neonatal rat cardiomycytes from A/R-induced injury in a chloride-dependent manner.

Objective:To investigate the significance of Th1/Th2 cytokines in prognostic stratification of acute myeloid leukemia (AML).Methods:A total of 83 patients with newly diagnosed AML from June 2017 to April 2019 in the First People's Hospital of Yunnan Province were collected. According to the Chinese guidelines for diagnosis and treatment of adult acute myeloid leukemia (non-acute promyelocytic leukemia) (2017 edition), AML patients were divided into poor prognosis group (45 cases), moderate prognosis group (19 cases), and good prognosis group (19 cases); moderate prognosis plus poor prognosis was treated as the not good prognosis. Mann-Whitney U test and Kruskal-Wallis H test were used to compare the expression differences of Th1/Th2 cytokines in peripheral blood of different prognosis groups; cytokines with statistical differences among different prognosis groups were selected, and the cut-off value of AML patients with different prognostic stratification distinguished by cytokines was determined by using receiver operating characteristic (ROC) curve. Finally, patients were divided into ≥ cut-off value group and 0.05).The cut-off value of TNF-β was 3.23 pg/ml in good prognosis group and moderate prognosis group, the area under the ROC curve was 0.866 (95% CI 0.753-0.978, P < 0.05); among 26 patients with TNF-β≥ 3.23 pg/ml, 25 (96.2%) patients had not good prognosis. The cut-off value was 3.62 pg/ml for distinguishing between moderate prognosis group and poor prognosis group, the area under the ROC curve was 0.747 (95% CI 0.610-0.884, P = 0.02); 18 (100%) patients with TNF-β≥ 3.62 pg/ml had not good prognosis. The cut-off value was 2.19 pg/ml for distinguishing between good prognosis group and not good prognosis group, the area under the ROC curve was 0.719 (95% CI 0.595-0.842, P = 0.04); among 53 patients with TNF-β≥2.19 pg/ml, 46 (86.8%) patients had not good prognosis. Conclusions:The high expression of TNF-β may indicate that the prognosis of AML patients is not good. When the level of TNF-β was equal or greater than 3.62 pg/ml, it may contribute to the prognostic stratification of AML patients.

Objective:To observe the therapeutic efficacy and prognosis of daratumumab combined with chemotherapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT) followed by daratumumab and lenalidomide maintenance treatment for primary plasma cell leukemia (PCL).Methods:The clinical data of a patient with primary PCL admitted to the First People's Hospital of Yunnan Province in January 2020 were retrospectively analyzed, and relevant literatures were reviewed.Results:The patient was diagnosed with primary PCL and treated with daratumumab combined with BD (bortezomib + dexamethasone) for 1 course and BCDD (bortezomib + cyclophosphamide + liposomaldoxorubicin + dexamethasone) for two courses. The patient was treated with daratumumab combined with allo-HSCT after complete remission. The donor cells were successfully implanted and the chimerism rate of donor cells was 94.36% without acute graft-versus-host disease reaction. And then the patient received intermittent maintenance therapy of daratumumab combined with low dose lenalidomide after transplantation, and the current remission period after transplantation reached 4 months.Conclusions:Daratumumab combined with chemotherapy bridging to allo-HSCT followed by daratumumab and lenalidomide may improve the prognosis of primary PCL and prolong survival time.