HLAⅠ基因型配合输注策略用于免疫性血小板输注无效的实验研究

目的利用血小板供者HLA基因分型库,针对PTR患者建立供受者HLA-A、B位点基因配合型输注策略,以提高临床血小板输注疗效。方法以血清学交叉配合试验作为免疫性PTR的初筛手段,对初筛阳性的35例PTR患者采取血小板基因配型策略,其中24例进行HLA-A、-B基因分型和血小板HLAⅠ类抗体检测,根据患者血小板基因型和供者特异性抗体(DSA)回避策略以及供受者CREG配合等级原则,在单采血小板因型数据库中搜寻配合型供者,其中DSA回避优先于CREG等级原则,共计83次输注。其余11例患者仅按照HLA交叉反应组CREG的配合等级原则选择供受者HLA-A,-B抗原基因型相合的供者,共计55次输注。回访临床患者相关资料及基因配型的血小板输注效果,统计分析校正的血小板计数增值(CCI)。结果对临床送检的35例PTR患者先后进行了453次ABO同型的血清学交叉配合试验,人均为12.94次(453/35),每次血小板交叉配型的供者平均人数为4.21(1908/453)人,血清学交叉配合试验阳性率为69.86%(1333/1908)。其中对24例患者标本进行了HLAⅠ类抗体检测,仅有1例标本为抗体阴性,即HLAⅠ类抗体阳性检出率为95.83%(23/24),且平均每个患者有(44.37±22.31)种特异性抗体;根据患者血清中抗体荧光强度值的高低,抗体为强阳性17例、阳性20例、弱阳性23例,其对应的检出率分别为73.91%(17/23)、86.96%(20/23)和100%(23/23)。138次HLA基因配合型输注后的血小板首次计数CCI均值为14.08±11.12[(23.95±21.28)h],高于1h CCI(>7.5有效)或24h CCI(>4.5有效),且DSA回避组输注效果(首次计数CCI为15.56±11.00)显著优于非DSA回避组(首次计数CCI为11.86±12.00)(P<0.05)。在所有患者输注中,49.28%的输注同时存在1种或多种非免疫因素。结论HLA-I基因配型血小板输注策略是预防和改善免疫相关性血小板输注无效的可行方法,对于多次输血且血小板相关抗体初筛阳性的患者,采取DSA回避和供受者CREG配合型输注策略可显著提高输血疗效,为临床提供血小板精准输注。

浙江省血小板供受者基因型配合信息系统构建及应用

血小板基因型配合为个体化精准输血提供了新途径。针对血小板基因库偏小、基因型配合流程信息化水平低的问题,浙江省血液中心构建了血小板供受者基因型配合信息系统。该系统汇集了全省血小板基因分型数据,功能覆盖血小板基因型配合闭环管理全流程,实现了全省献血者血小板基因分型数据共享。后续需加大宣传力度,提升匹配献血者招募效率;充分利用临床数据,持续优化血小板基因型配合流程;加强血小板供者基因型数据库建设,实现精准配型,丰富临床应用资源等。

Elimination Mechanism and Equilibrium Model about Complete Linkage of Panmixis Based on Fitness

[Objective]The aim was to discuss elimination mechanism and equilibrium model about complete linkage gene of panmixis based on fitness.[Method]A mathematical model that gsgenerational evalution of frequency of panmixis two pairs of complete linkage gene under difference fitness was built by using difference equations to study elimination mechanism and genetic equilibrium state of panncticcoinplete linkage gene.[Result]The panmixis is different from Hardy-Weinberg Equilibrium which reached the equilibrium just by one generation.A method that by using linear equation to calculate the frequency of four gametes and the fitnessed of panmixis was established after detormining is the fitness of 10 genotypes the necessary and sufficient condition of the panmixis to reach balance.[Conclusion]The method is valuable for reference on linkage disequilibrium and hitchhiking effect study.

Alloy gene Gibbs energy partition function and equilibrium holographic network phase diagrams of Au_3Cu-type sublattice system

Taking Au3Cu-type sublattice system as an example, three discoveries have been presented. First, the fourth barrier to hinder the progress of metal materials science is that today’s researchers do not understand that the Gibbs energy function of an alloy phase should be derived from Gibbs energy partition function constructed of alloy gene sequence and their Gibbs energy sequence. Second, the six rules for establishing alloy gene Gibbs energy partition function have been discovered, and it has been specially proved that the probabilities of structure units occupied at the Gibbs energy levels in the degeneracy factor for calculating configuration entropy should be degenerated as ones of component atoms occupied at the lattice points. Third, the main characteristics unexpected by today’s researchers are as follows. There exists a single-phase boundary curve without two-phase region coexisting by the ordered and disordered phases. The composition and temperature of the top point on the phase-boundary curve are far away from those of the critical point of the Au3Cu compound; At 0 K, the composition of the lowest point on the composition-dependent Gibbs energy curve is notably deviated from that of the Au3Cu compounds. The theoretical limit composition range of long range ordered Au3Cu-type alloys is determined by the first jumping order degree.

Alloy gene Gibbs energy partition function and equilibrium holographic network phase diagrams of AuCu_3-type sublattice system

Taking AuCu3-type sublattice system as an example, three discoveries have been presented: First, the third barrier hindering the progress in metal materials science is that researchers have got used to recognizing experimental phenomena of alloy phase transitions during extremely slow variation in temperature by equilibrium thinking mode and then taking erroneous knowledge of experimental phenomena as selected information for establishing Gibbs energy function and so-called equilibrium phase diagram. Second, the equilibrium holographic network phase diagrams of AuCu3-type sublattice system may be used to describe systematic correlativity of the composition?temperature-dependent alloy gene arranging structures and complete thermodynamic properties, and to be a standard for studying experimental subequilibrium order-disorder transition. Third, the equilibrium transition of each alloy is a homogeneous single-phase rather than a heterogeneous two-phase, and there exists a single-phase boundary curve without two-phase region of the ordered and disordered phases; the composition and temperature of the top point on the phase-boundary curve are far away from the ones of the critical point of the AuCu3 compound.

Fiber-modified adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene from the human telomerase reverse transcriptase promoter induces apoptosis in human hepatocellular carcinoma cells

AIM: Because of a major resistance to chemotherapy, prognosis of hepatocellular carcinoma (HCC) is still poor. New treatments are required and gene therapy may be an option. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in multiple malignant tumors, and using adenoviral vectors has shown a targeted tumor-specific therapy. However, repeated administration of adenoviral vectors can lead to cell resistance, which may be caused by the initial coxsackie-adenovirus receptor (CAR). One technique to overcome resistance is the use of modified adenoviral vectors containing an Arg-Gly-Asp (RGD) sequence. In this study we constructed an adenoviral vector (designated Ad/TRAIL-F/RGD) with RGD-modified fibers, expressing the TRAIL gene from the human telomerase reverse transcriptase (hTERT) promoter, and evaluated its antitumor activity in HCC cell lines.METHODS: To investigate the effects of Ad/TRAIL-F/RGD in human HCC cell lines Hep G2 and Hep 3b, cells were infected with Ad/CMV-GFP (vector control), Ad/gTRAIL (positive control), and Ad/TRAIL-F/RGD. Phosphatebuffered saline (PBS) was used as control. Cell viability was determined by proliferation assay (XTT), and apoptosis induction by fluorescence activated cell sorting (FACS).RESULTS: Cells treated with Ad/TRAIL-F/RGD and Ad/ gTRAIL showed a significantly reduced cell viability in comparison to PBS and Ad/CMV-GFP treatment in both cell lines. Whereas, treatment with PBS and Ad/CMVGFP had no cell-killing effect. The reduced cell viability was caused by induction of apoptosis as shown by FACS analysis. The amount of apoptotic cells was similar after incubation with Ad/gTRAIL and Ad/TRAIL-F/RGD. CONCLUSION: The new RGD modified vector Ad/TRAILF/RGD could become a potent therapeutic agent for the treatment of HCC, adenovirus resistant tumors, and CAR low or negative cancer cells.

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronichepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope.RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining.CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.

Mechanism of counterattack of colorectal cancer cell by Fas/Fas ligand system

AIM： To determine the role of Fas/Fas ligand （FasL） in the immune escape of colon cancer cells. METHODS： Immunohistochemistry was used to observe the expression of Fas and FasL in the tissues of colon cancer patients. In situ hybridization was used to detect the localization of FasL mRNA expression in cancer tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling （TUNEL） assay and CD45 staining were performed to detect the apoptosis of tumor-infiltrating lymphocytes （TILs）. Co-culture assays of colon cancer cells （SW480） and Jurkat cells （Fas-sensitive cells） were performed to observe the counterattack of colon cancer cells to lymphocytes. RESULTS： Of 53 cases of colon carcinomas, 23 cases （43.4%） expressed Fas which was significantly lower as compared to the normal colonic mucosa （73.3%, P〈0.01）, and 45 cases （84.9%） of colon carcinomas expressed FasL, whereas only two cases （3.75%） in normal mucosa expressed FasL. FasL expression in the colon cancer cells was found to be associated with increased cell death of TIEs. The apoptotic rate of TIL in the FasL-positive staining regions of tumor cells was significantly higher than that in the FasL-negative staining region （54.84±2.79% vs 25.73±1.98%, P〈0.01）. The co-culture of SW480 cells and Jurkat cells confirmed the function of FasL on the SW480 cells. The apoptotic rates of Jurkat cells were found to be related with the amount of SW480 cells. CONCLUSION： Colon cancer cells can escape the immune surveillance and killing via decreasing Fas expression, and can counterattack the immune system via increasing FasL expression. Fas/FasL can serve as potential targets for effective antitumor therapy.

Overexpression of decoy receptor 3 in hepatocellular carcinoma and its association with resistance to Fas ligand-mediated apoptosis

AIM： To characterize the expression and genomic amplification of decoy receptor 3 （DcR3） in hepatocellular carcinoma （HCC） and to evaluate the role of DcR3 in apoptosis.METHODS： We examined 48 cases of HCC for DcR3 expression by RT-PCR and DcR3 gene amplification by quantitative genomic PCR. DcR3 protein was detected by immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick and labeling （TUNEL） was used to identify the apoptosis cells in tissues. Primary hepatoma cell culture and MTT test were used to evaluate the protection against FasL- and chemicalinduced apoptosis by DcR3 expression. RESULTS： DcR3 mRNA overexpression was detected in 60% HCC （29/48） patients. The occurrence of HCC was not associated with amplification of the gene. One sample base substitution was found in three sites as a sequence in Genbank. The expression of DcR3 in HCC was associated with the apoptotic index （0.067±0.04 vs 0.209±0.12, P〈0. 01）, size of mass, stage, and infiltration or metastasis （41.2% vs71.0%, 40% vs75%, 51.8% vs84.6%, P〈0. 05）. DcR3 expression could protect hepatoma cells against apoptosis induced by FasL, but not by chemicals. CONCLUSION： These data suggest that in addition to gene amplification there may be another mechanism underlying DcR3 overexpression. The effect of overexpression of DcR3 on the apoptosis of cancer cells may have direct therapeutic implications for the management of HCC.

Tumor necrosis factor-related apoptosis-inducing ligand gene on human colorectal cancer cell line HT29

AIM: To evaluate the therapeutic efficiency of Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) gene on human colorectal cancer cell line HT29.METHODS: Human embryonal kidney cells transformed by introducing sheared fragments of Ad5 DNA (293 cell)were used for amplification of adenoviral vectors: Ad/GTTRAIL,Ad/GT-Bax, Ad/GT-LacZ and Ad/PGK-GV16. Human colorectal cancer cell line HT29 was transfected with binary adenovirus-mediated TRAIL gene. Bax gene was used as positive control, LacZ gene was used as the vector control,and cells treated with PBS only were used as a mock control. The morphological changes, cell growth and apoptosis were measured by reversmicroscope, MTT method and flow cytometry.RESULTS: All adenoviral vectors titer determined by optical absorbency at A260nm were 1×1010 viral particle/ml(vp/ml).Obviously morphological changes of HT29 cells were observed when infected with Ad/GT-TRAIL, and these changes were much more obviously when Ad/PGK-GV16was coinfected. The cell suppression percentage and the percentage of apoptotic cells were 52.5 % and 16.5 %respectively when infected with Ad/GT-TRAIL alone, while combining with Ad/PGK-GV16, the growth of HT29 was suppressed by 85.2 % and the percentage of apoptotic cells was 35.9 %. It showed a significantly enhanced therapeutic efficiency with binary system (P＜0.05).CONCLUSION: A binary adenoviral vector system provides an effective approach to amplify viral vectors that express potentially toxic gene, TRAIL. Ad/GT-TRAIL showed a significantly enhanced therapeutic efficiency for HT29 when coinfected with Ad/PGK-GV16. Ad/GT-TRAIL could induce apoptosis of HT29 and inhibit its growth.

Expressions of chemokine receptor CXCR4 and its ligand CXCL12 in salivary adenoid cystic carcinoma

Objective: To examine expressions of chemokine receptor CXCR4 and its ligand CXCL12 in primary focus and lymphogenous metastasis of salivary adenoid cystic carcinoma (ACC) with lung metastasis. Methods: Using immunohistochemical hypersensitivity catalyzed signal amplification (CSA), expressions of chemokine receptor CXCR4 and ligand CXCL12 were detected in tissue specimens from 20 cases of primary cancer focus and lymphogenous metastasis of salivary adenoid cystic carcinoma, of which 7 cases were associated with lung metastasis and 3 with lympogenous metastasis. Twenty cases of tongue carcinoma (including 10 cases with lymphogenous metastasis) and 15 cases of mucoepidermoid carcinoma (including 5 cases with lymphogenous metastasis) were used as the malignant control group;and salivary mixed tumor (n=10), tongue leukoceratosis (n=10) and cervical lymph node reactive hyperplasia (n=10) were used as the benign control group. Results: Expression of CXCR4 in the tissues and lymph metastases of oral and maxillofacial salivary ACC, mucoepidermoid carcinoma and tongue carcinoma was significantly higher than that of the benign control group (P<0.05); expression of CXCR4 in the primary focus of ACC was significantly higher than that of the malignant control group; and expression of CXCR4 in the ACC with lung metastasis was 87.1% (6/7), significantly higher than that without lung metastasis(P<0.01). There was evident positive expression of CXCL12 in endotheliocytes of microvessels within cancer and paracancer tissues and significantly high expression of CXCL12 in lymphogenous metastasis(P<0.05). Conclusion: Chemokine receptor CXCR4 and its ligand CXCL12 may be associated with local invasion and lymphogenous metastasis of oral and maxillofacial cancer, especially with lung metastasis of salivary ACC.

Indole methylation protects diketopiperazine configuration in the maremycin biosynthetic pathway

The maremycin biosynthetic gene cluster has been identified in Streptomyces sp. B9173. Comparative metabolic profiling with knockout mutant strains led to the identification of new products correlated to the maremycin biosynthesis, in particular the ＂demethyl＂-maremycins with an unexpected D-tryptophan unit. A biosynthetic pathway for the maremycins is proposed and plausible reasoning for tryptopban epimerization in the demethylmaremycin biosynthesis is also provided.

Roles of flanking sequences in the binding between unimolecular parallel-stranded G-quadruplexes and ligands

G-quadruplexes attract more and more attention in recent years.Numerous small molecules which can induce or stabilize the formation of G-quadruplexes have been investigated on the purpose of anticancer drug development.As a motif existed in physiological condition,flanking sequences are an important part of G-quadruplexes but the study on the impact of flanking sequences on (G-quadruplex)-ligand binding is rarely reported.In this paper,the effects of flanking sequences on binding affinity between a series of unimolecular parallel-stranded G-quadruplex sequences derived from c-myc oncogene promoter (termed as c-myc G-quadruplexes) and their ligands are discussed in detail.The results showed that the flanking sequences on c-myc G-quadruplexes play key roles in (G-quadruplex)-ligand interaction.When a c-myc G-quadruplex is bound to its ligands,the flanking sequences might form a binding cavity above the terminal G-quartet,which could provide a suitable site for ligands to dock in.Moreover,the bases on flanking sequences could interact with ligand through π-π stacking,and finally form a sandwich-stacking mode (terminal G-quartet,ligand and bases on the flanking sequence).This mode could stabilize the (G-quadruplex)-ligand complex effectively and enhance the binding affinity dramatically.However,flanking sequences are also found to exhibit steric hindrance effect which could impede the (G-quadruplex)-ligand binding.