[Clinical Application of Preoperative Autologous Blood Donation in Selective General Surgery].

OBJECTIVE: To study the clinical effects of preoperative autologous blood donation (PABD) in selective general surgery. METHODS: Paired study was performed in PABD group with 70 PABD cases screened from selective general surgery during the period from November 2017 to August 2018 in our hospital, and the control group included 70 cases without preoperative autologous blood donation, the baseline data before surgery were not significantly different. The transfusion quantities of allogeneic RBC and plasma, the levels of perioperative hemoglobin and platelets, the time and expense of hospitalization were compared between two groups. RESULTS: The levels of Hb and Plt in PABD group before and after blood collection were determined as follows: 138.26±14.73 g/L vs 127.52±13.36 g/L (P＜0.05) and (221.67±52.86)×109/L vs (198.35±52.65)×109/L (P＞0.05) respectively. The analysis of allo-RBC and allo-plasma transfusion in PABD group and control group showed that: the quantity of allogeneic RBC transfusion was 0.20±0.71 U and 0.89±0.97 U, and the quantity of allogeneic plasma transfusion was 30.43±100.81 ml and 106.52±152.61 ml (P＜0.05) respectirdy during perioperation. The comparison results of preoperative Hb and plt in PABD group and control group were 135.65±14.16 g/L vs 134.15±11.98 g/L and (270.36±58.28)×109/L vs (271.67±65.02) ×109/L respectively. The levels of postoperative Hb and plt in PABD group and control group were 120.24±14.40 g/L vs 121.20±14.30 g/L at 1 d after operation, and (241.80±63.58)×109/L vs (241.30±69.11)×109/L at 1 d after operation respectively; 123.15±13.80 g/L vs 121.65±14.33 g/L at 3 d after operation and (251.26±72.94)×109/L vs (255.54±73.85)×109/L at 3 d after operation; 122.78±13.92 g/L and 122.00±13.82 g/L (before discharge) and (262.50±80.96)×109/L and (264.56±71.08)×109/L (before discharge, platelet). These data were not statistically different (P＞0.05). The hospitalization time was 14.84±3.37 days and 14.84±2.24 days, respectively, without statistical difference (P＞0.05) in two groups. The expenses of hospitalization and the blood transfusion in two groups were 50627.27±9889.45 RMB and 50979.43±8195.00 RMB; 354.39±362.57 RMB and 684.02±425.53 RMB (P＜0.05). CONCLUSION: The application of PABD reduces the use of allogeneic blood and costs for patients undergoing selective surgery with blood losts of 1000 ml.

[Effect of 2Me, DTT, ZAAP and Enzyme on JMH Antigen on the Surface of Human Erythrocytes].

OBJECTIVE: To explore the the effects of 2-Me, DTT, papain, pineapple protease and ZZAP on the antigenicity of JMH antigen of human red blood cells (RBC) surface. METHODS: Firstly, human RBC were treated with 2-Me, DTT, pineapple protease, papain and ZZAP reagents, respectively. The antigenicity of JMH antigen on human RBC surface was detected and analyzed by flow cytometry. RESULTS: Flow cytometric analysis found that compared with level before treatment, the antigenicity of JMH antigen on RBC surface was significantly reduced after 2-Me treatment, the positive rate of JMH antigen: 69.5%±4.5% vs 56.5%±3.4% (t=12.44, P＜0.01); fluorescence intensity: 4906±317 vs 3003±165 (t=11.84, P＜0.01). The antigenicity of JMH antigen on RBC surface significantly increased after DTT treatment, showing the positive rate of JMH antigen: 61.7%±3.8% vs 75.5±4.9% (t=16.57, P＜0.01), fluorescence intensity: 4044±294 vs 4854±319 (t=15.46, P＜0.01). However, both bromelain and papain could significantly reduce the antigenicity of JMH antigen on the RBC surface, Bromelain: the positive rate of JMH antigen: 62.2%±3.8% vs 8.8%±1.2% (t=26.44, P＜0.01), fluorescence intensity: 4263±273 vs 1444±212 (t=19.27, P＜0.01); Papain: the positive rate of JMH antigen: 62.8%±3.6% vs 8.8%±1.5% (t=21.38, P＜0.01), fluorescence intensity: 4389±284 vs 1458±230 (t=17.49, P＜0.01). The flow cytometric analysis revealed that ZZAP treatment significantly reduced the antigenicity of JMH antigen on the RBC surface, the positive rate of JMH antigen: 62.2%±4.4% vs 48.2%±4.1% (t=14.87, P＜0.01), fluorescence intensity: 4106±263 vs 2063±175 (t=17.49, P＜0.01). CONCLUSION: The treatment with 2-Me can reduce the antigenicity of JMH antigen on human RBC surface. The antigenicity of JMH antigen on human RBC surface increased after DTT treatment. The antigenicity of JMH antigen on human RBC surface significantly reduces after the treatment with pineapple protease or papain. ZZAP treatment can reduce the antigenicity of JMH antigen on the RBC surface.

[Determination and correlation analysis of contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot].

OBJECTIVE: To determine and perform a correlation analysis of the contents of putrescine, cadaverine, and histamine in necrotic tissue, blood, and urine of patients with diabetic foot (DF). METHODS: Ten patients with severe wet necrotizing DF hospitalized from January 2011 to January 2012 were assigned as group DF, and 10 orthopedic patients with scar but without diabetes or skin ulcer hospitalized in the same period were assigned as control group. Samples of necrotic tissue from feet of patients in group DF and normal tissue from extremities of patients in control group, and samples of blood and 24-hour urine of patients in both groups were collected, and the amount of each sample was 10 mL. Contents of putrescine, cadaverine, and histamine were determined with high performance liquid chromatography-mass spectrometry. The data got from the determination of blood and urine were processed with t test, and those from necrotic or normal tissue with Wilcoxon rank sum test. The correlation of contents of polyamines between necrotic tissue and blood, blood and urine were processed with simple linear regression analysis. RESULTS: (1) Contents of putrescine, cadaverine, and histamine in the necrotic tissue of group DF were (186.1 ± 26.8), (78.553 ± 12.441), (33 ± 10) mg/kg, which were significantly higher than those in normal tissue of control group [(2.2 ± 1.2), (1.168 ± 0.014), 0 mg/kg, with Z values respectively -3.780, -3.781, -4.038, P values all below 0.01]. The content of putrescine in necrotic tissue of group DF was significantly higher than those of cadaverine and histamine (with Z values respectively -3.780, -3.630, P values all below 0.01). (2) Contents of putrescine, cadaverine, and histamine in the blood of group DF were (0.075 ± 0.013), (0.022 ± 0.003), (0.052 ± 0.014) mg/L, and they were significantly higher than those in the blood of control group [(0.014 ± 0.009), (0.013 ± 0.003), (0.016 ± 0.008) mg/L, with t values respectively 6.591, 2.207, 3.568, P < 0.05 or P<0.01]. The content of putrescine in the blood of group DF was significantly higher than those of cadaverine and histamine (with t values respectively 13.204, 3.096, P values all below 0.01). (3) Contents of putrescine, cadaverine, and histamine in the urine of group DF were (0.735 ± 0.088), (0.450 ± 0.012), (0.1623 ± 0.0091) mg/L, and only the contents of putrescine and cadaverine were significantly higher than those in the urine of control group [(0.050 ± 0.014), (0.035 ± 0.007) mg/L, with t values respectively 3.270, 4.705, P<0.05 or P<0.01]. The content of putrescine in the urine of group DF was significantly higher than that of cadaverine (t = 6.686, P < 0.01). (4) There were significant and positive correlations in contents of putrescine, cadaverine, and histamine between necrotic tissue and blood in patients of group DF (with r values respectively 0.981, 0.994, 0.821, P values all below 0.01). There were no significant correlations in contents of putrescine, cadaverine, and histamine between blood and urine in patients of group DF (with r values respectively 0.150, 0.239, 0.177, P values all above 0.05). CONCLUSIONS: Putrescine, cadaverine, and histamine exist in the necrotic tissue of patients with DF in high concentrations, among which putrescine predominates. These polyamines can be absorbed into the blood through wound and excreted through the urine.

Nur-related receptor 1 gene polymorphisms and alcohol dependence in Mexican Americans.

AIM: To investigate the association of polymorphisms of nur-related receptor 1 (Nurr1) and development of alcohol dependence in Mexican Americans. METHODS: Peripheral blood samples were collected from 374 alcoholic and 346 nonalcoholic Mexican Americans; these two groups were sex- and age-matched. Sample DNA was extracted and genomic DNA was amplified by polymerase chain reaction. The -2922(C) 2-3 polymerase chain reaction products were digested with Sau96I, alleles of 1345(G/C), and -1198(C/G) in the regulatory region as well as Ex+132 (G/T/A/C) and Ex+715(T/-) in exon 3 were studied by sequencing. RESULTS: The C2/C2, C2/C3, C3/C3 genotype distribution of -2922(C) 2-3 was 34.4%, 38.2% and 27.5% in the nonalcoholic group compared to 23.3%, 51.2% and 25.4% in the alcoholic group (P = 0.001). The C/C, C/G, G/G genotype distribution of -1198(C/G) was 23.5%, 46.1% and 30.3% in the nonalcoholic group compared to 13.9%, 50.9% and 35.3% in the alcoholic group (P = 0.007). However, the -1345 (G/C), Ex3+132(G/T/A/C) and Ex3+715(T/-) alleles were not polymorphic in Mexican Americans, and all those studied had G/G, G/G and T/T genotype for these three alleles, respectively. The -2922(C) 2-3 did not show allele level difference between alcoholic and nonalcoholic individuals, but -1198 (C/G) showed a significant allele frequency difference between alcoholic (39.3%) and nonalcoholic (46.6%) populations (P = 0.005). Excluding obese individuals, significant differences were found at both genotypic and allelic levels for the -2922(C) 2-3 polymorphism (P = 0.000 and P = 0.049) and the -1198 (C/G) polymorphism (P = 0.008 and P = 0.032) between nonobese alcoholics and nonobese controls. Excluding smokers, a significant difference was found only at the genotypic level for the -2922(C) 2-3 polymorphism (P = 0.037) between nonsmoking alcoholics and nonsmoking controls, but only at the allelic level for the -1198(C/G) polymorphism (P = 0.034). CONCLUSION: Polymorphisms in the regulatory region of Nurr1 are implicated in pathogenesis of alcohol dependence and the Nurr1/dopamine signaling pathway might be important for this dependence development in Mexican Americans.

Establishment of a transplantable human myeloid BALB/c nude mouse model.

To establish a mouse model bearing transplantable human chronic myeloid leukemia for hematopoietic stem cell transplantation to treat leukemia, 4 - 5-week-old female BALB/c nude mice were given cyclophosphamide 2 mg/mouse at day -2, -1, and then the human chronic myeloid leukemia K562 cells were engrafted into the mice at day 0 by injection via tail vein or peritoneal cavity. PB and BM cells were collected, the CD45, CD13, and CD33 antigens were delected by using FCM, the bcr/abl fusion gene mRNA was examined by RT-PCR. The results showed that transplantable leukemic mice could be yielded from 4 - 5-week-old nude mice either by injection through tail vein or peritoneal cavity when the total number of inoculated tumor cells was more than 2 x 10(5) per mouse, whether being pretreated with 2 mg CTX/mouse or not. The transplanted mice could survive 30 - 60 day with leukemia. In conclusion, the mouse model bearing leukemia can be established by inoculation 2 x 10(5) K562 cells into immunodeficient BALB/c nude mice.

Ex vivo expansion of T, NK and CD34+ cells from umbilical cord blood.

Umbilical cord blood stem cell transplantation (CBSCT) has made significant progress in treatment of lethal congenital or malignant disorders. Both the incidence and severity of GVHD from CBSCT were lower than that from bone marrow and peripheral blood stem cell transplantation, particularly for adult patients, but these advantages were also associated with higher rates of relapse. The immune-mediated effect of natural killer and cytotoxic T cells against residual tumor cells were shown to prevent relapse and to induce remission after bone marrow transplantation. To explore possibility of ex vivo expansion of T, NK and CD34(+) cells from umbilical cord blood, cord blood was expanded ex vivo with different combinations of cytokines, T and NK cells proliferation and differentiation were observed. CB MNCs were separated in Ficoll-Isopaque column and cultured in IMDM for 14 days with different recombinant cytokines. Cultured cells were collected and analyzed for progenitor/stem cell immunophenotyping at day 0, 3, 7, and 14 by using flow cytometry. The results indicated that all test groups cultured with different combinations of SCF, IL-3, IL-6, IL-7, IL-2 showed significant expansion of UCB MNC, compared with the group without cytokines. All test groups showed expansion effects on CD34(+) cells, CD34(+) percentage went up from 1.6% in fresh CB to the highest 11.9% in group D (SCF + IL-3, IL-6, IL-2). The CD34(+) cells peak displayed at day 7 of culture in group A and D, while in other two groups B and C appeared at day 14 of culture. The expansion multiple of CD34(+) cells in all test groups at day 7 of culture were from 10 to 50. The average value of CD3(+) T cell in fresh UCB was 18.7 +/- 4.3%, the CD3(+) T cells decreased sharply in the medium without any interleukin, while obvious increase were observed in the other test groups containing different combinations of cytokines. The maximal expansion multiple of CD3(+) T cells reached 2 times of the fresh UCB level. CD56(+) cells amounted to 3.6 +/- 1.9% of fresh UCB, CD56(+) cell number increased significantly only in medium containing IL-2. It is concluded that T cells, NK cells as well as stem/progenitor cells can be expanded in the same time from CB-MNC with the combinations of cytokines.

In vitro effects on proliferation, telomerase activity and apoptosis of an eremophilanoid sesquiterpene from Senecio oldhamianus maxim in cultured human tumor cell lines.

8,11-Dioxol-6-en-9alpha, 10alpha-epoxy-8beta-hydroxyeremophilane (HEM), a new eremophilanoid sesquiterpene, was isolated from Senecio oldhamianus Maxim. Its effects of cytotoxicity, telomerase activity, apoptosis and related genes expression in two human tumor cell lines, human hepatoma cells SMMC-7721 and human oophoroma cells HO-8910, were studied. Hydroxycamptothecine (HCPT) was used as a positive control. The IC50 of cytotoxicity by HEM were 24.9 +/- 2.1 and 19.4 1.6 microM in SMMC-7721 and HO-8910 cells respectively, and 0.35 +/- 0.10 and 0.27 +/- 0.08 microM for HCPT. HEM inhibited telomerase activity with the IC50 35.9 +/- 3.2 microM in SMMC-7721 and 25.6 +/- 2.6 microM in HO-8910 cells, while HCPT had no effect on telomerase activity in both tumor cell lines. HEM 20-30 microM induced apoptosis in SMMC-7721 cells from 5.7% to 18.4% and in HO-8910 cells from 7.6% to 67.1%. While HCPT 0.1-0.5 microM induced apoptosis in SMMC-7721 cells from 6.5% to 13.3% and in HO-8910 cells from 9.9% to 30.9%. HEM 30 microM significantly decreased Bcl-2 protein expression to 58.7% in SMMC-7721 and to 57.6% in HO-8910 cells. While HCPT 0.5 microM significantly decreased Bcl-2 protein expression to 64.3% in SMMC-7721 and to 70.0% in HO-8910 cells. HEM 25 microM and 30 microM significantly increased P53 protein expression 2.3-3.6-fold in SMMC-7721 and 3.0-5.7- fold in HO-8910 cells. While HCPT 0.5 microM significantly increased P53 protein expression 3.3-fold in SMMC-7721 and 2.7-fold in HO-8910 cells. Overall, HCPT exhibited a more potent effect on cytotoxicity and apoptosis in the two tumor cell lines than HEM did. However HEM can inhibit telomerase activity in the two tumor cell lines but HCPT cannot.

[Apoptosis and regulation of expressions of apoptosis-related gene Bcl-2 and p53 induced by selenium dioxide in three leukemia cell lines].

OBJECTIVE: To investigate the mechanisms underlying the effect of selenium dioxide (SeO(2)) on the proliferation, apoptosis, and apoptosis-related gene expressions of Bcl-2 and p53 in 3 leukemia cell lines NB4, K562 and HL-60. METHODS: The three leukemia cell lines were treated with 3, 10 and 30 mmol/L SeO(2) and apoptosis detected by flow cytometry and analysis of p53 and Bcl-2 expressions. RESULTS: SeO(2) at 10 and 30 mmol/L could inhibit the proliferation of three leukemia cell lines. SeO(2) treatment at 30 mmol/L for 48 h induced an apoptosis rate of 54.0 %, 46.5 %, 49.6 % in NB4, K562, and HL-60 cells respectively, and down-regulated Bcl-2 expression in NB4 and K562 but not in HL-60 cells. CONCLUSION: SeO(2) can induce apoptosis in NB4, K562 and HL-60 leukemia cells, involving the down-regulation of Bcl-2 and up-regulation of p53.

[Method of detection of soluble HLA-I and soluble HLA-I level alteration in storage blood].

Aim of this study was to develop the detection method of soluble human leukocyte antigens I (sHLA-I) and to explore sHLA-I level alteration in storage blood and its significance. sHLA-I level in sera of 60 Guangdong normal individuals and sHLA-I concentration in blood components from 20 donors quantitatively were detected by sandwich ELISA. The results showed that sensitivity of this assay was 2.84 ng/ml. Coefficients of variation were 5.80% within assays and 9.00% between assays respectively. The recovery rate was >/= 98.57%. The sHLA-I level of normal individuals in Guangdong was (699.54 +/- 360.10) ng/ml. sHLA-I in red blood cells stored for 28 days and in random-donor platelets were significantly higher than that in other blood components and their amount was proportionate to the number of residual donor leukocytes and to the length of storage. In conclusion, sandwich ELISA assay for detection of sHLA-I is a sensitive, specific and stable technique. Blood components with different concentration of sHLA-I may be chosen for clinical transfusion.

Allogeneic peripheral blood hematopoietic stem cell transplantation in malignant hematopoietic diseases.

To evaluate the use of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) for treatment of acute and chronic leukemia, from March 1997 to January 2003, 21 adult patients with malignant hematopoietic diseases underwent allo-PBSCT from HLA-identical siblings (19 patients) and haplo-identical mother (one) and one B point site mismatched sibling (one). All donors were mobilized with G-CSF for 4 days and peripheral blood stem cells were collected by CS-3000 separator. The conditioning regimen included the high dose combination chemotherapy and TBI. Cyclosporine-A (CsA) plus a short course of MTX was used for GVHD prophylaxis in all patients. The results showed that after trans plantation, median time for the recovery of granuocyte > or = 0.5 x 10(9)/L and platelets > or = 20 x 10(9)/L were 12 (10 - 20) and 15 (11 - 35) days, respectively. Acute GVHD was observed in 8/17 patients (47%), of which one transplanted from HLA-haploidentical mother. Chronic GVHD occurred in 12/17 patients (70%). All of four female survivals did not show acute and chronic GVHD. Day 100 transplantation-related mortality was 14% (3/21). Relapse occurred in two patients (9.5%) who underwent allo-PBSCT in stage of non-remission at one and six months. After follow-up of 40 (15 - 70) months, 11 patients (52.4%) are still disease-free survival. These results suggested that peripheral blood stem cells produce a faster hematopoietic recovery and a lower relapse of leukemia. The rate of aGVHD is not increased when using the peripheral blood as source of stem cells; however, cGVHD continues to be a significant problem. Donors tolerated the procurement procedure without complications.

[Observation on gene polymorphism of Rh blood group in Chinese Han nationality].

To observe the gene polymorphism of Rh blood group in unrelated random individuals and families for Chinese Han nationality, polymerase chain reaction-sequence specific primer (PCR-SSP) was used to amplify the Rh C/E gene, RhD gene, exons, intron 2 and 10, insert and Rh Box in 160 blood samples of RhD positive unrelated individuals and 71 samples of RhD negative unrelated individuals and 7 samples of families whose probands were RhD-negative. The results showed that RhD genes of RhD-negative individuals with C antigens were polymorphism, three forms were found for D exon including intact, partial deletion and complete deletion exons. Insert fragments and Rh Box were found in most cases of families whose probands were RhD-negative and its inheritance accorded with the Mendel's Law, and it did not affect the expression of RhD gene. "Normal" RhD exon 4 amplifying product was not found in all of the samples. It was concluded that gene structure of the RhD-negative in Chinese was polymorphism, intact, partial deletion and complete deletion exons were found in the individuals with C antigen and probably existed specific D (nf) Ce haplotype. The function of insert was uncertain. The Rh gene sequences of Chinese Han nationality are different from those of Caucasian and the Rh gene library based on Han nationality should be established.

[Telomerase Activity Expressed in Acute Leukemia Cell]

To explore the possible linkage between telomerase and acute leukemia, we detected telomerase activity expressed in 3 leukemia cell lines, 22 acute leukemia bone marrow and 6 normal bone marrow with PCR ELISA assay. Results showed that telomerase activities of three leukemia cell lines were positive with the average (1.57 +/- 0.056) U, normal bone marrow samples average was (0.085 +/- 0.081) U, telomerase value from 22 acute leukemia patients was (0.512 +/- 0.294) U. Telomerase activity is higher expressed in acute leukemia than normal samples and decreased significantly after chemotherapy (P < 0.01). The results suggested that telomerase activity was related to some malignant diseases and might be used as a marker for tumor diagnosis and therapy.