SOX12基因在Vero细胞感染猪流行性腹泻病毒过程中的功能研究

[目的]探究SOX12基因对猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)复制的影响,以期为抗PEDV育种提供有效分子标记。[方法]本研究合成3条SOX12基因干扰序列(siRNA1、siRNA2、siRNA3)及其阴性对照(siNC)并转染至非洲绿猴肾细胞(Vero细胞),转染48 h后收集细胞,利用实时荧光定量PCR检测干扰效率。将有效的干扰片段和siNC分别转染至Vero细胞24 h后,用感染复数为0.05的PEDV感染细胞,分为4组:感染PEDV 12 h试验组(12 hpi-siRNA)、感染PEDV 12 h对照组(12 hpi-siNC)、感染PEDV 24 h试验组(24 hpi-siRNA)及感染PEDV 24 h对照组(24 hpi-siNC),利用实时荧光定量PCR检测PEDV N及SOX12基因的表达水平,通过Western blotting检测各组细胞PEDV N蛋白表达水平,利用组织半数感染量(50%tissue culture infective dose, TCID_(50))检测PEDV的病毒滴度,并利用间接免疫荧光(indirect immunofluorescence assay, IFA)法检测各组细胞中PEDV复制情况。通过GeneMANIA网站预测SOX12基因的互作基因,利用实时荧光定量PCR检测抑制SOX12基因表达后其互作基因的表达变化。[结果]SOX12基因3条干扰片段的干扰效率为30%~60%,其中siRNA3干扰效率最高,可达60%。与12 hpi-siNC和24 hpi-siNC组相比,12 hpi-siRNA和24 hpi-siRNA组PEDV N基因的转录和蛋白表达水平均显著降低(P<0.05)。病毒滴度表型测定结果表明,12 hpi-siRNA和24 hpi-siRNA组的PEDV病毒滴度显著低于各自对照组(P<0.05);IFA结果也表明,12 hpi-siRNA和24 hpi-siRNA组的PEDV复制明显少于对照组。基因互作预测及实时荧光定量PCR检测结果显示,与siNC组相比,干扰SOX12基因的表达后,其互作基因SOX17、DDX51、ZMIZ2、SSRP1、TAF6和ABCB8的表达水平均显著降低(P<0.05)。[结论]本研究揭示了SOX12基因在PEDV感染Vero细胞过程中的调控作用,发现SOX12基因的下调表达能显著抑制PEDV复制和其互作基因SOX17、DDX51、ZMIZ2、SSRP1、TAF6和ABCB8的表达。

RDH12-associated retinal degeneration caused by a homozygous pathogenic variant of 146C>T and literature review

AIM:To describe the clinical,electrophysiological,and genetic features of an unusual case with an RDH12 homozygous pathogenic variant and reviewed the characteristics of the patients reported with the same variant.METHODS:The patient underwent a complete ophthalmologic examination including best-corrected visual acuity,anterior segment and dilated fundus,visual field,spectral-domain optical coherence tomography(OCT)and electroretinogram(ERG).The retinal disease panel genes were sequenced through chip capture high-throughput sequencing and Sanger sequencing was used to confirm the result.Then we reviewed the characteristics of the patients reported with the same variant.RESULTS:A 30-year male presented with severe early retinal degeneration who complained night blindness,decreased visual acuity,vitreous floaters and amaurosis fugax.The best corrected vision was 0.04 OD and 0.12 OS,respectively.The fundus photo and OCT showed bilateral macular atrophy but larger areas of macular atrophy in the left eye.Autofluorescence shows bilateral symmetrical hypo-autofluorescence.ERG revealed that the amplitudes of a-and b-wave were severely decreased.Multifocal ERG showed decreased amplitudes in the local macular area.A homozygous missense variant c.146C>T(chr14:68191267)was found.The clinical characteristics of a total of 13 patients reported with the same pathologic variant varied.CONCLUSION:An unusual patient with a homozygous pathogenic variant in the c.146C>T of RDH12 which causes late-onset and asymmetric retinal degeneration are reported.The clinical manifestations of the patient with multimodal retinal imaging and functional examinations have enriched our understanding of this disease.

Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes

A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

香蕉枯萎病菌ste12基因的克隆与序列分析

为了解ste12基因在尖孢镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖孢镰刀菌古巴专化型1号和4号生理小种之间的致病力差异关系,采用PCR和RT-PCR方法扩增了2个生理小种的ste12基因,并对扩增产物进行了测序及相似序列搜索和比对,还对基因编码的蛋白进行了氨基酸序列比对和分析。研究结果表明,2个生理小种ste12基因开放阅读框均为2070 bp,存在7个碱基的差异,基因同源性为99.7%。编码689个氨基酸,氨基酸序列一个有差异。根据生物信息学软件预测编码蛋白没有信号肽,具有两个相同的功能位点,分子量和PI分别为75 ku和6.47。

新生隐球菌STE12α基因的克隆及表达载体的构建

目的从新生隐球菌的基因组中扩增出STE12α基因,并构建相应的表达载体,以进一步研究STE12α基因对隐球菌的生长特性及致病性的影响。方法采用PCR方法以及基因重组方法扩增并克隆新生隐球菌基因组中的STE12α基因,建立具有表达野生型STE12α基因的表达载体。结果从新生隐球菌的基因组获得STE12α全基因,建立重组子pUCm-STE12α/NovaBlue以及重组表达载体质粒pGAPZ-STE12α,实现了STE12α基因的转化并获得表达。结论成功地克隆了新生隐球菌STE12α基因并构建了可表达野生型STE12α基因的表达载体,为进一步研究STE12α基因功能打下了良好的基础。

Ste12基因对玉米大斑病菌渗透胁迫的调控作用

前期研究表明,玉米大斑病菌Ste12基因对分生孢子发育和致病性有重要的调控作用。本试验利用Ste12基因的RNAi沉默突变体StRNAi 9-10和StRNAi 3-6分析该基因对玉米大斑病菌渗透胁迫的调控能力。通过比较野生型菌株和RNAi沉默突变体在0.4mol/L CaCl2、1mol/L KCl、1mol/L NaCl、1mol/L山梨醇等渗透胁迫条件下的菌落形态、生长速度、菌丝形态、产孢量等指标,发现StRNAi9-10对1 mol/L NaCl和1mol/L KCl胁迫的耐受能力显著增强,对1mol/L CaCl2胁迫的耐受能力显著降低,对1mol/L山梨醇的耐受力无显著差异;StRNAi 3-6对4种胁迫条件的耐受能力均显著降低。以上研究结果表明,Ste12基因不仅调控玉米大斑病菌的分生孢子发育和致病力形成,而且参与玉米大斑病菌的渗透胁迫调控。

STE12α基因对新生隐球菌形态学影响的初步研究

目的研究STE12α基因对新生隐球菌形态学的影响。方法分别敲除血清A型和血清B型新生隐球菌菌株的STE12α基因,建立缺陷株,再将STE12α基因重新导入建立重建株。观察并比较野生株、STE12α基因缺陷株及重建株在体内、外孵育后菌落和菌落的形态学差异。结果 STE12α基因缺陷株组形成的菌落明显偏少,菌株直径偏小,荚膜发育不良,而重构株组这些方面的改变都得到了恢复。结论 STE12α基因对新生隐球菌的形态学改变有着重要的影响,可能直接影响其毒力。

Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

Construction of retroviral vector of p^(125FAK) specific ribozyme genes and its effects on BGC-823 cells

AIM： To construct the retroviral vector of p^125FAK specific ribozyme genes and to explore the feasibility of ribozyme in BGC-823 gene therapy in vitro. METHODS： A hammerhead ribozyme DNA targeting p^125FAK mRNA from nt 1010 to nt 1032 was synthesized and recombinated into the retroviral vector pLXSN forming pLRZXSN recon. Using the lipofectin-mediated DNA transfection technique, pLRZXSN was introduced into BGC-823 cells. The effects of ribozyme on the growth of BGC-823 cells and apoptosis were studied by cell colony assay, flow cytometry （FCM）, reverse transcriptasepolymerase chain reaction （RT-PCR）, detection of DNA fragmentation and electron microscopy. RESULTS： The number of BGC-823 cell colonies was inhibited by 56% after the cells were treated for 48 h. The cell proliferation was inhibited effectively by p^125FAK ribozyme and the inhibitory effect depended on the concentration and the time of incubation. The expression of p^125FAK mRNA and protein p^125 decreased sharply in BGC-823 cells treated with p^125FAK ribozyme. The characteristics of apoptosis, namely sub-G1 peak, DNA fragmentation and morphological changes, were revealed in BGC-823 cells treated with p^125FAK ribozyme. CONCLUSION： p^125FAK ribozyme decreases p^125FAK gene expression and induces apoptosis of human gastric cancer cells in vitro.

THE INFLUENCE OF HUMAN SINGLE CHAIN INTELEUKIN-12 GENE TRANSDUCTION ON THE BIOLOGICAL BEHAVIOR OF HEPATOMA 7721 CELLS

Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL- 12 cells which secrete hscIL- 12 stably, were obtained via G418 selection, and in vitro the influence of hscIL- 12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL- 12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice,respectively. 7721/pcDNA and 7721/hscIL- 12 groups were divided into two sub- groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL- 12 produced stably by 7721/hscIL- 12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL- 12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL- 12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5～ 7mm,while those of 7721/hscIL- 12 group were 2～ 4mm.When treated with hPBL, the tumor of 7721/hscIL- 12 group disappeared completely. Histologically, the tumors from 7721/hscIL- 12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis , whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. Conclusion. hscIL- 12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL- 12 has certain effects on mice immune system. These findings suggest that hscIL- 12 and hscIL- 12 gene therapy might have promising prospects in clinical application.

Frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among children with sensorineural deafness in China

Objective： To investigate the frequency of mitochondrial 12S rRNA gene A1555G and 961 insC mutations among Chinese with sensorineural deafness. Methods： Blood samples from 78 sporadic cases with sensorineural deafness were obtained and DNA was extracted from the leukocytes, then the mitochondrial DNA target fragments were amplified by polymerase chain reaction（PCR）. The 1555G mutations were detected by BsmA 1 restriction endonuclease digestion, every fragment was analyzed by sequencing; All the 961 insC mutation were detected by direct sequencing. Results： The percent age of A1555G mutation and mt961C insertion were 6.4% and 2.6% in the hearing-impaired Chinese subjects respectively. Conclusion： A1555G and 961insC mutations in mitochondrial DNA 12S rRNA gene regions may play a role in the pathogenesis of hearing loss in the sporadic cases.

Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L.ssp.chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.

Combination therapy of murine liver cancer with IL-12 gene and HSV-TK gene

Objective: To investigate the synergistic anti-tumor effects of murine IL-12 gene and HSV-TK gene therapy in mice bearing liver cancer. Methods: Mouse liver cancer MM45T. Li (H-2d) cells were transfected with retroviral vector containing IL-12 gene or HSV-TK gene insert. Gene-modified liver cancer cells, MM45T. Li/IL-12 and MM45T. Li/TK, with stable expression of IL-12 and TK were obtained. Balb/c mice were inoculated subcutaneously with 2′105 MM45T. Li cells. When the tumor reached a size of 0.5-1.0 cm, a mixture of MM45T.Li/TK cells and 60Co-irradiated MM45T. Li/IL-12 cell were injected intratumoraly. Ganciclovir (GCV) was injected ip (40 mg.kg-1.d-1) for 10 days. Intratumoral injection of 60Co-irradiated MM45T. Li/IL-12 cells was repeated twice in one week apart. Mice with distant tumors were treated according to the same protocol. CTL activity of spleen cells was measured by 51Cr-release assay and phenotype of tumor infiltrating lymphocytes by immunohistochemical staining. Results: In mice treated with MM45T. Li/IL-12 or MM45T. Li/TK+GCV individually led to moderate reduction in tumor growth, but neither could eradicate the tumor completely, while in 60% of mice treated with a mixture of MM45T. Li/IL-12 and MM45T. Li/TK cells plus GCV, complete tumor regression was observed, with no tumor recurrence for two months. The growth of distant tumor was also inhibited significantly in mice similarly treated. Most of the mice received combined gene therapy plus GCV had abundant CD4+, CD8+T lymphocyte infiltration. Their CTL activity was significantly higher than in mice received single gene therapy. Conclusion Combination therapy with IL-12 gene and HSV-TK gene plus GCV is effective for mouse liver cancer.

A phylogeny of the Passerida(Aves: Passeriformes)based on mitochondrial 12S ribosomal RNA gene

Background: Passerida is the largest avian radiation within the order Passeriformes. Current understanding of the high-level relationships within Passerida is based on DNA–DNA hybridizations; however, the phylogenetic relationships within this assemblage have been the subject of many debates.Methods: We analyzed the 12 S ribosomal RNA gene from 49 species of Passerida, representing 14 currently recognized families, to outline the phylogenetic relationships within this group.Results: Our results identified the monophyly of the three superfamilies in Passerida: Sylvioidea, Muscicapoidea and Passeroidea. However, current delimitation of some species is at variance with our phylogeny estimate. First, the Parus major, which had been placed as a distinct clade sister to Sylvioidea was identified as a member of the super family;second, the genus Regulus was united with the Sturnidae and nested in the Muscicapoidea clade instead of being a clade of Passerida.Conclusion: Our results were consistent with Johansson's study of the three superfamilies except for the al ocation of two families, Paridae and Regulidae.

The Effect of Polymorphisms of MTHER Gene and Vitamin B on Hyperhomocysteinemia

The relationship between hyperhomocysteinemia and coronary artery disease (CAD) was investigated and the influence of environmental factors (Folate, VitB12) and genetic factors [N5,N10 methylenetetrahydrofolate reductase gene (MTHFR) or MTHFR gene mutation] on plasma homocysteine (Hcy) levels and the risk of CAD observed. Fifty one CAD patients and 30 CAD free subjects were recruited in the study. The polymorphisms of MTHFR gene were analyzed by PCR RFLP and plasma total Hcy levels were measured by high performance liquid chromatography with fluorescence detection. Plasma folate and vitamin B12 concentrations were measured by an automated chemiluminescence method. It was found that mean total plasma Hcy concentrations were significantly higher in CAD patients than in CAD free subjects ( P <0.01). The differences were also apparent among the three genotypes of MTHFR gene in CAD group ( P <0.05). There was no significant difference in the genotype distributions and allele frequencies between the two groups. A strong inverse correlation was found between folate or vitamin B12 and plasma Hcy levels according to MTHFR genotype ( P <0.01). It was concluded that hyperhomocysteinemia is a new independent risk factor for CAD. However, MTHFR gene mutation alone does not relate significantly to the morbidity of CAD since hyperhomocysteinemia and its influence on the risk of CAD are decided by both environmental and genetic factors. Supplementary treatment with vitamins B can effectively lower the plasma levels of Hcy, thus maybe reduceing the risk of CAD.

Novel compound heterozygous mutation of SLC12A3 in Gitelman syndrome co-existent with hyperthyroidism:A case report and literature review

BACKGROUND Gitelman syndrome(GS)is a rare inherited autosomal recessive tubulopathy,characterized clinically by hypokalemia,hypomagnesemia,hypocalciuria,and metabolic alkalosis,and is caused by an inactivating mutation in SLC12A3.GS is prone to misdiagnosis when occurring simultaneously with hyperthyroidism.It is important to consider the possibility of other diseases when hyperthyroidism is combined with hypokalemia,which is difficult to correct.CASE SUMMARY A female patient with hyperthyroidism complicated with limb weakness was diagnosed with thyrotoxic hypokalemic periodic paralysis for 4 mo.However,the patient’s serum potassium level remained low despite sufficient potassium replacement and remission of hyperthyroidism.GS was confirmed by whole exome and Sanger sequencing.Gene sequencing revealed compound heterozygous mutations of c.488C>T(p.Thr163Met),c.2612G>A(p.Arg871His),and c.1171_1178dupGCCACCAT(p.Ile393fs)in SLC12A3.Protein molecular modeling was performed to predict the effects of the identified missense mutations.All three mutations cause changes in protein structure and may result in abnormal protein function.All previously reported cases of GS coexisting with autoimmune thyroid disease are reviewed.CONCLUSION We have identified a novel compound heterozygous mutation in SLC12A3.The present study provides new genetic evidence for GS.

血浆lncRNA SNHG12、miR-133b预测乳腺癌新辅助治疗效果的价值

目的探讨血浆长非编码RNA小核仁宿主基因12(lncRNA SNHG12)、微小RNA(miR)-133b预测乳腺癌新辅助治疗效果的价值。方法选取2022年1月至2023年1月在该院进行新辅助治疗的86例乳腺癌患者作为乳腺癌组,依据新辅助治疗效果分为病理学完全缓解(pCR)组和non-pCR组。另选取同期在该院进行健康体检的50例健康女性作为对照组。检测并比较所有研究对象lncRNA SNHG12、miR-133b表达水平。采用多因素Logistic逐步回归模型分析影响乳腺癌新辅助治疗效果的因素。绘制受试者工作特征(ROC)曲线分析血浆lncRNA SNHG12、miR-133b单独及联合检测对乳腺癌新辅助治疗效果的预测价值。结果乳腺组血浆lncRNA SNHG12表达水平高于对照组,miR-133b表达水平低于对照组,差异均有统计学意义(P<0.05)。pCR组纳入34例患者,non-pCR组纳入52例患者。pCR组与non-pCR组雌激素受体、孕激素受体、人表皮生长因子受体-2(HER-2)、细胞增殖核抗原、分子分型比较,差异均有统计学意义(P<0.05)。pCR组lncRNA SNHG12表达水平低于non-pCR组,miR-133b表达水平高于non-pCR组,差异均有统计学意义(P<0.05)。多因素Logistic逐步回归分析结果显示,分子分型为HER-2过表达型、lncRNA SNHG12表达水平升高、miR-133b表达水平降低是乳腺癌新辅助治疗效果的危险因素(P<0.05)。ROC曲线分析结果显示,血浆lncRNA SNHG12、miR-133b单独检测预测乳腺癌患者新辅助治疗效果的曲线下面积(AUC)分别为0.850、0.951,均低于二者联合检测的0.963。86例乳腺癌中Luminal A型11例(pCR 1例、non-pCR 10例)、Luminal B型43例(pCR 12例、non-pCR 31例)、HER-2过表达型16例(pCR 14例、non-pCR 2例)、三阴型16例(pCR 7例、non-pCR 9例)。ROC曲线分析lncRNA SNHG12、miR-133b对4种不同亚型乳腺癌新辅助治疗效果的预测效能结果显示,血浆lncRNA SNHG12、miR-133b单独及联合检测预测Luminal B型乳腺癌患者新辅助治疗效果的AUC分别为0.753、0.974、0.981,预测HER-2过表达型乳腺癌患者新辅助治疗效果的AUC分别为0.804、0.857、0.893。预测三阴型乳腺癌患者新辅助治疗效果的AUC分别为0.849、1.000、1.000。结论乳腺癌患者血浆lncRNA SNHG12表达增高,miR-133b表达降低,二者联合检测对乳腺癌新辅助治疗效果具有较好的预测价值。

外源添加VB_(12)对肺炎克雷伯氏菌代谢3-羟基丙酸的影响

为了探究外源添加维生素B12(Vitamin B12,VB_(12))对Klebsiella pneumoniae HD79和K.pneumoniae HD79-T利用甘油还原途径生产3-羟基丙酸(3-hydroxypropionic acid,3-HP)的影响以及摸索VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值上限,将不同浓度(0.01、0.02、0.03、0.04、0.05 g/L)的VB_(12)添加到K.pneumoniae HD79及K.pneumoniae HD79-T的发酵培养基中,利用HPLC检测其底物消耗及产物产生情况、qRT-PCR检测还原途径相关基因的mRNA表达情况以及酶联免疫试剂盒检测代谢相关酶活性。结果表明,VB_(12)对K.pneumoniae HD79和K.pneumoniae HD79-T的阈值为0.01 g/L和0.03 g/L。与未添加VB_(12)相比,菌株K.pneumoniae HD79和K.pneumoniae HD79-T的3-HP产量分别提高了24.39%,8.86%;醛脱氢酶基因puuC表达量分别提高了2.49倍和1.68倍;ALDH、GDHt和PDOR的酶活力分别提高了50.24%、40.36%和18.29%,及30.49%、37.84%和13.56%。说明通过外源添加辅酶因子VB_(12)对肺炎克雷伯氏菌高产3-HP是可行策略。