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The expression level of metabolitic enzyme (CYP1A1, CYP1B1) and its correlation with cell differentiation /apoptosis were studied as well. The influence of resveratrol in cell cycle was determined with FCM. Results 1 Resveratrol not only suppresses the growth of medulloblastoma cells in a dose and time related fashion but also induces cell differentiation and apoptosis. Resveratrol promotes differention of UW228-1,-2 cells to forward glia, UW228-3 and Med-3 cells to neuron. A treatment of resveratrol in the concentration of 100μM for 48hrs comit most of cells die of apoptosis. 2 Among the four cell lines Fas and Caspase-3 were constantly expessed, whereas FasL was absent irrespective to the drug treatment. 3 ICC, RT-PCR, Western-blot hybridization and EROD enzymatic activity assay demonstrated that expression of CYP1A1 is different after treatment with resveratrol in four cell lines, up-regulated in three UW228 cell lines in a dose dependent manner but down-regulated in Med-3 cells. Suppressive effect of resveratrol on CYP1B1 expression is the same among four medulloblatoma cell lines. 4 Cell cycle distribution of UW228-3 was greatly changed after treatment.

结果:1、白藜芦醇以时间和剂量依赖性方式抑制髓母细胞瘤细胞生长,同时促进细胞的分化和凋亡:白藜芦醇促使UW228-1、-2靶细胞向神经胶质细胞方向成熟分化,UW228-3、Med-3细胞向神经元细胞方向成熟分化,以100μM作用48小时效果显著;2、四个细胞系均有Fas和Caspase-3表达,但无Fas-L基因表达和蛋白活性产生,因此难以形成Fas相关性死亡通路;3、白藜芦醇处理前后CYP1A1基因的表达在各细胞系略有不同:UW228-1,-2,-3三个细胞系白藜芦醇以剂量相关性方式上调CYP1A1的表达,而Med-3细胞系,白藜芦醇却抑制CYP1A1的表达;四个细胞系在白藜芦醇处理后CYP1B1的表达均受到抑制;4、UW228-3细胞系经白藜芦醇作用后细胞周期有较大改变,表现在G0/G1期在整个细胞周期中所占的比例增加,而其它时期则相应减少。

The living cell of L428 cell has experienced from monoclonal to the multi-cell forming process,and presents a big cell around many small clone cell encystation phenomenon.finally,gigantic cells is dead in the life time.3.The cell counting result showed that the proportion of big cell or the H/RS type cell(diameter≥25um)is for(11.6±1.5)%in the L428 group,for(4.6±0.7)%in L428-MVC group and for(13.1±1.3)%in L428-EVC group,respectively.

经持续稀释成单个大细胞、单个小细胞的L428细胞,小细胞可分裂转化成大细胞,大细胞亦可生成小细胞;常见单个大细胞周围出现多个小细胞围绕的现象,在此过程中NF-kB一直持续活化。3。

Fig.1 SHEE cultured on coverslide, the living cells were growing in single layer with rich cytoplasm, the nuclei were uniform in size with a nucleolus ph ×400 Fig.2 SHEE had a nucleus with ellipse shape, large nucleolus and the cytoplasm contained mitochondria and tonofibrilEM ×10 000 Fig.3 SHEE was spherical in shape, with pseudopods attached on petri dish and abundant villi on cell surface SEM ×5 000 Fig.4 Same as in Fig.3, cell attached on petri dish, appeared stellate or polygonal in shape, with abundant pseudopods and cytoplasmic processes. Protrusive nuclear region in central part of the cell had more micro-villi SEM ×5 000 Fig.5 Chromosomes of SHEE cells belonged to diploidy type Giemsa ×1 000 Fig.6 The SHEE cells of stained in dark brown by Ki67 immunohistochemistry were the proliferative cells Immunohistochemistry ×400 Fig.7 In SHEE cell culture, the nucleus stained red or pink by PI was dead cell, the green nucleus was living cell Fluorescent ×400 Fig.8 The cell labeled by TdT was apoptotic cell in which the chromatin of nucleus condensed in block, a pyknotic nucleus in the upper right conner was seen TdT labeled ×400

图1 SHEE培养在盖坡片上,活细胞单层生长,胞浆较丰富,细胞核大小一致,有核仁×400 图2 SHEE培养细胞细胞核椭圆形,核仁较大,胞浆有较丰富的线粒体和张力原纤维EM ×10 000 图3 SHEE细胞呈球状,有伪足贴壁,表面有密集微绒毛SEM ×5 000 图4 同上细胞贴壁,呈星状或多角形,有丰富伪足和胞浆突,核区隆起有较多微绒毛SEM ×5 000 图5 SHEE细胞染色体仍属二倍体Giemsa染色×1 000 图6 SHEE细胞Ki67免疫组织化学染棕黄色为增殖细胞×400 图7 SHEE培养细胞出现死细胞,胞核和胞浆PI染色呈红色或淡红色,蓝色细胞核为活细胞荧光显微镜×400 图8 细胞TdT标记阳性为凋亡细胞,染色质凝集呈块状,右上角有一固缩细胞核TdT标记×400

The animal model of this method system have, the characteristics of the high fat, high leptin, high Ins, and match with the characteristic of clinical and the pathologic of simple obesity. This studies showed: The acupuncture can obviously reduce the vaule of intaking food and water of the fat rats, and lower the body weight and fat; The acupuncture can urge the diameter, area, physical volume of white fat cell contract, Brown fat tissue of fat rat group surroundings vascular containing large quantity of white fat cell, the acupuncture group was less. this showed: The acupuncture enhanced the white fat cell metabolism, and promotesed the white fat cell to convert to brown fat cell; The frequency of spontaneous electric of nerve cell of pvn of the fat rat is high, the acupuncture counld depress the spontaneous electric of nerve cell of pvn, This showed: the acupuncture can depress the appetite passing to adjust function of pvn nerve; The monoaminergic neurotransmitter of inside of the fat rat brain is disfunction, the acupuncture can rectify this disfunction, and adjust level of 5-HT, and affect appetite, and improve metabolism; The level of leptin and Ins of fat rat increased high, The level of it of acupuncture group decreased, compared with the fat group p.001, This showed: the fat rats have the defeat of leptin and Ins in the body, The acupuncture can lowering this defeat, The level of leptin and Ins of the fat rat in the brain is less than the normal group, The level of leptin of the acupuncture group is high than the fat group, This showed: the acupuncture can increase the transportation of leptin from blood to brain.

本方法所制肥胖大鼠模型具有高体重、高脂、高leptin和高Ins血症的特点,符合人类单纯性肥胖病的临床和病理特征;实验研究显示:针灸能明显减少肥胖大鼠的摄水、摄食量及降低其体重、体脂;针灸能促使白色脂肪细胞直径、面积、体积缩小,减少肥胖大鼠棕色脂肪组织血管周围白色脂肪细胞数量,显示:针灸增强了白色脂肪细胞的代谢,促进了白色脂肪细胞向棕色脂肪细胞的转化:肥胖大鼠PVN神经细胞自发放电频率增加,脑内单胺递质紊乱,针灸通过抑制PVN亢奋的自发放电,调整5-HT水平,达到影响食欲,改善代谢的作用;肥胖大鼠血中leptin及Ins水平增高,与正常组大鼠比有明显差异,提示大鼠体内存在leptin及Ins抵抗,针灸后肥胖大鼠随着体重体脂的下降,血中leptin、Ins水平降低,与肥胖组比均有显著性差异,而针灸后大鼠脑内leptin增加,与肥胖组比P.05,提示:针灸具有改善leptin、Ins抵抗及增加leptin脑内转运的作用。

Comparing the landscape types of 1995 with that of 2005, we find out that:①the natural arbors are decreasing, with patches more fragmentation, size more odds, shapes more complex;②the area of natural shrubberies is increasing, with patches less fragmentation, shapes more simple and formulary ,the size of patches more even;③the area of natural grasses is increasing, with patches less fragmentation, shapes of patches more odds;④the area of timbers is increasing, with patches more dominance in landscape, more connectivity;⑤the area of Eucalyptus increases, with patches less fragmentation, shapes of patches more complex , more convergence, more connectivity;⑥the area of orchards is increasing, more connectivity;⑦the patches of the rubber is becoming more assemble, more connectivity, more formulary;⑧the area of plowland increases;⑨the area of reservoirs, rivers and ponds increases, with patches more assemble;⑩the area of cities decreases, with patches more dispersing, with shape more complexthe area of countries increases, with patches less connectivity, more dispersing, with shape more complex; the patches of the rest lands are more dispersing, with shape more complex;(3)In the process of economic development in the upper reaches of the Changhuajiang river watershed, a series of the ecological environment problems have appeared because a lot of landscape function broke off, and the connection of human landscape with natural landscape decreased, which aroused by the fragmentation of natural and semi-nature landscapes increased, the stability of the landscape structure decreased, as well as stress of human disturbance, resource and environment upon the landscape increased gradually.

3昌化江上游流域在经济发展进程中,自然与半自然景观破碎度增加,自然景观结构稳定性下降,人为干扰、资源和环境对景观胁迫逐渐增大,中断了许多生态过程,导致城市等人文景观与自然景观连接度很差,产生了一系列的生态环境问题。

In the brain of adult rat, the positive immunohistochemical product of lSL-l (ISL-l-positive) was mainly located in the neuronal nucleus and found in discrete regions except to brain cortex, such as the Purkinje cell layer and the granular cell layer of cerebellum, the granular cell layer and the pyramidal cell layer of hippocampus, the mitral cell layer, the internal and external plexiform layer, the granular cell layer and the granular cell layer of olfactory bulb and so on, and several nuclei of the hypothalamus, midbrain and pons, such as claustrum, anterior olfactory nucleus, accumbens nucleus, caudate-ptamen, pallidum, substantia nigra, striatum, islands of Callaje, mammillary nucleus, anterior pretactal nucleus, habenular nucleus, amygdaloid nucleus, cuneate nucleus, rubral nucleus, gigantocellular reticular nucleus and so on.

在正常成年大鼠脑中,同源框基因islet-1表达产物(ISL-1)免疫组织化学阳性物质广泛分布于除大脑皮层外的神经细胞的细胞核内,ISL-1阳性神经元密集分布于小脑Purkinje细胞层和颗粒细胞层、海马的颗粒细胞层和锥体细胞层、嗅球的内丛层、外丛层、颗粒细胞层及僧帽细胞层等,另外在丘脑、中脑和桥脑的一些重要神经元核团均有分布,如,屏状核、前嗅核、伏核、尾壳核、苍白球、黑质、纹状体、Calleja岛、乳头体核、前顶盖前核、缰核、杏仁核、楔束核、红核网状巨细胞核等。

The present studies showed that two cell populations were found in haemocytes: large cell with high granularity and small cell with low granularity by flow cytometry FCM on light scanttering pattern. Two distinct cell types were identified based on phase contrast microscope: one type of cell was dark and dioptric aberration, while the other was bright and dioptric strong. By Giemsa and H.E staining, cytoplasmic staining were heterogeneous and internal particles were obvious in one type of cell, while cytoplastic staining were homogeneous and internal particles were inexistent in the other type of cell. By transmission electron microscope, we found that the mitochondria, Golgi apparatus organelles were rich and internal particles were obvious in one type of cells, and contrary to the another cells.

流式细胞术光散射图谱显示血细胞被分两类,一类为颗粒度高的大细胞,另外一类为颗粒度低的小细胞;相差显微镜观察显示,血细胞可分为胞体暗、折光性差和胞体明亮、折光性强的两类; Giemsa和H.E染色显示细胞分为胞质染色不均一、胞内颗粒明显和胞质染色均一、胞内颗粒不明显的两类;透射电镜超薄切片观察显示,颗粒明显的细胞胞质内线粒体、高尔基体等细胞器较丰富,颗粒不明显的细胞胞质内细胞器较少;负染结果表明血细胞主要分为表面不光滑、突起明显和细胞表面光滑、突起较不明显的两类。

The stable clones are further identified by RT-PCR and Western blot; 6 MTT assay is used to investigate the effect of ZNRD1 on the cell growth of cells (AGS, SGC7901, MKN28, NIH3T3, GES-1); 7 Soft agar assay is used to investigate the effect of ZNRD1 on the clonality of cells (AGS, MKN28); 8 Nude mice assay is used to investigate the effect of ZNRD1 on the cell growth of gastric cancer cells (AGS, MKN28); 9 Flow cytometry is used to investigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1); 10 Flow cytometry is used to investigate the effect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3); 11 MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitro; 12 SRCA is used to investigate the effect of ZNRD1 on the drug sensitivity of gastric cancer cells (SGC7901, SGC7901/VCR) in vivo; 13 Flow cytometry is used to investigate the effect of ZNRD1 on adriamycin accumulation of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR); 14 Transmission electron microscope is used to investigate the effect of ZNRD1 on the sensitivity of SGC7901 cells towards drug-induced apoptosis; 15 Flow cytometry and DNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivity of cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-induced apoptosis; 16 Microarray is used to investigate the profiling of ZNRD1-responsive genes in gastric cancer cells (AGS, MKN28, SGC7901, SGC7901/VCR); 17 RT-PCR and Western blot are used to identify the results of microarray; 18 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of cyclin D1; 19 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of MDR1; 20 Kinase assay is used to investigate the effect of ZNRD1 on the activity of cyclin E-CDK2 kinase; 21 The antisensenucleic acids of p21 is used to inhibit the expression of p21, and flow cytometry is used to investigate the effect of p21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 22 The antisensenucleic acids of p27 is used to inhibit the expression of p27, and flow cytometry is used to investigate the effect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 23 Liposome is used to up-regulate the expression of Skp2, and flow cytometry is used to investigate the effect of Skp2 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 24 Western blot is used to investigate the effect of ZNRD1 on the stability of Skp2 and p27 in gastric cancer cells; 25 MVD assay is used to investigate the effect of ZNRD1 on the angiopoietic activity of gastric cancer cells; 26 ELISA is used to investigate the effect of ZNRD1 on the expression of VEGF165 in gastric cancer cells; 27 The roles of DARPP-32 in MDR of gastric cancer cells are investigated using gene transfection, MTT assay, SRCA, flow cytometry and DNA ladder assay.

应用杂交瘤技术制备ZNRD1的首个单克隆抗体;2)利用RT-PCR、Western blot和免疫组化检测ZNRD1在胃癌组织、胃炎组织、正常胃上皮组织、胃癌细胞和正常胃组织上皮细胞中的表达;3)构建ZNRD1的小干扰RNA载体,并测序鉴定;4)利用脂质体将ZNRD1的真核表达载体及其空载体转染胃癌细胞(AGS、SGC7901、MKN28)和小鼠成纤维细胞(NIH3T3),G418筛选后进行鉴定;5)利用脂质体将ZNRD1的小干扰RNA载体及其空载体转染药敏胃癌细胞(SGC7901)、正常胃组织上皮细胞(GES-1)、对长春新碱耐药的胃癌细胞(SGC7901/VCR)、药敏白血病细胞(HL-60)、对长春新碱耐药的白血病细胞(HL-60/VCR),G418筛选后进行鉴定;6)利用MTT实验检测ZNRD1高/低表达对细胞(AGS、SGC7901、MKN28、NIH3T3、GES-1)生长的影响;7)通过软琼脂克隆形成实验检测上调ZNRD1对AGS、MKN28细胞克隆形成能力的影响;8)通过裸鼠成瘤实验检测上调ZNRD1对AGS、MKN28细胞体内成瘤性的影响;9)通过流式细胞仪分析ZNRD1高/低表达对细胞(AGS、MKN28、NIH3T3、GES-1)的细胞周期的影响;10)通过流式细胞仪分析上调ZNRD1对细胞(AGS、MKN28、NIH3T3)的凋亡的影响;11)通过MTT实验检测ZNRD1高/低表达对细胞(SGC7901、SGC7901/VCR、HL-60、HL-60/VCR)体外药物敏感性的影响;12)通过肾包膜下移植法检测ZNRD1高/低表达对细胞(SGC7901、SGC7901/VCR)体内药物敏感性的影响;13)通过流式细胞仪分析ZNRD1高/低表达对细胞(SGC7901、SGC7901/VCR、HL-60、HL-60/VCR)内阿霉素蓄积和泵出的影响;14)通过透射电镜检测上调ZNRD1对SGC7901细胞凋亡敏感性的影响;15)通过流式细胞仪和DNA梯度试验检测ZNRD1高/低表达对细胞(SGC7901、SGC7901/VCR、HL-60)凋亡敏感性的影响;16)通过基因芯片检测ZNRD1高/低表达对胃癌细胞内基因表达谱的影响;17)利用RT-PCR、Western blot对基因芯片的结果进行鉴定;18)利用报告基因实验检测ZNRD1对cyclin D1的启动子活性的调节作用;19)利用报告基因实验检测ZNRD1高/低表达对MDR1的启动子活性的调节作用;20)利用激酶试验检测ZNRD1对cyclin E-CDK2 激酶活力的影响;21)利用反义核酸技术抑制p21的表达;通过流式细胞仪检测抑制p21对ZNRD1介导的细胞周期阻滞的影响;22)利用反义核酸技术抑制p27的表达;通过流式细胞仪检测抑制p27对ZNRD1介导的细胞周期阻滞的影响;23)利用脂质体转染法上调Skp2的表达;通过流式细胞仪检测上调Skp2对ZNRD1介导的细胞周期阻滞的影响;24)利用Western blot检测ZNRD1对p27和Skp2的蛋白稳定性的影响;25)利用微血管密度实验检测ZNRD1对AGS、MKN28细胞裸鼠移植瘤微血管形成的影响;26)利用ELISA检测ZNRD1对AGS、MKN28细胞培养上清和移植瘤匀浆中VEGF165含量的影响;27)利用脂质体转染法、MTT实验、肾包膜下移植法、流式细胞仪和DNA梯度试验检测新耐药相关分子DARPP-32对细胞(SGC7901、SGC7901/VCR、对阿霉素耐药的胃癌细胞SGC7901/ADR)多药耐药表型的影响;利用脂质体转染法和MTT实验检测下调ZNRD1对DARPP-32介导的胃癌多药耐药的调控作用。

E studied expression levels of WAVE1 in six leukemia cell lines (the human Jurkat T leukemia cells, U937 histiocytic lymphoma cells, Burkitts lymphoma cell Raji, acute promyelocytic leukemia cell HL-60, chronic myelogenous leukemia cell K562 and K562/A02) by Western blotting analysis. Levels of WAVE1 expression were high in all six human leukemia cancer cell lines. In contrast, the constitutive expression levels of WAVE1 were noticeably lower in normal human peripheral blood mononuclear cells, or non-blood cancer cell-lines, including human lung A549 cancer cells, Hela cervical cancer cells, MG-63 osteosarcoma cells, CNE2 nasopharyngeal carcinoma cells, human umbilical vein endothelial cell, QSG7701 hepatocarcinoma cells, and WI-38 lung fibroblastoma cells.

AVE1在人类血液肿瘤细胞系(人T细胞白血病细胞系Jurkat、人组织细胞淋巴瘤细胞系U937、人Burkitts淋巴瘤细胞系Raji、人原髓细胞白血病细胞系HL-60、人慢性髓原白血病细胞系K562和K562/A02)中高表达,在非血液肿瘤细胞系(人肺腺癌细胞系A549、人宫颈癌细胞系Hela、人成骨肉瘤细胞系MG-63、人鼻咽癌细胞系CNE2、人脐静脉内皮细胞系HUVEC、人肺成纤维细胞系WI-38、人肝上皮细胞系QSG7701)以及正常人外周血单个核细胞中低表达或不表达。

Trans-Blot Cells and Systems 170-3825 Trans-Blot Cell With Wire Electrodes and PowerPac HC Power Supply, 100120/220240 V, includes 2 gel holder cassettes, buffer tank, lid with power cables, 4 fiber pads, 1 pack precut blot absorbent filter paper (15 x 20 cm), power supply, power cord, instructions 170-3850 Trans-Blot System With Plate Electrodes and PowerPac HC Power Supply, 100120/220240 V 170-3853 Trans-Blot System With Plate Electrodes, Super Cooling Coil, and PowerPac HC Power Supply, 100120/220240 V, includes 2 gel holder cassettes, buffer tank, lid with power cables, 4 fiber pads, 1 pack precut blot absorbent filter paper (15 x 20 cm), power supply, power cord, instructions 170-3910 Trans-Blot Cell With Wire Electrodes, includes 2 gel holder cassettes, buffer tank, lid with power cables, 4 fiber pads, 1 pack precut blot absorbent filter paper (15 x 20 cm) 170-3939 Trans-Blot Cell With Plate Electrodes and Super Cooling Coil, includes 2 gel holder cassettes, buffer tank, lid with power cables, 4 fiber pads, 1 pack precut blot absorbent filter paper (15 x 20 cm) 170-3946 Trans-Blot Cell With Plate Electrodes, includes 2 gel holder cassettes, buffer tank, lid with power cables, 4 fiber pads, 1 pack precut blot absorbent filter paper (15 x 20 cm) Trans-Blot Cell Accessories 170-3912 Super Cooling Coil, required for all high-intensity transfers 170-3913 Gel Holder Cassette, includes 2 fiber pads 170-3914 Fiber Pads, 15.5 x 20.5 cm, 6 170-3920 Trans-Blot Standard Wire Electrode Card, cathode 170-3921 Trans-Blot Standard Wire Electrode Card, anode 170-3922 Trans-Blot Cell Buffer Tank 170-3923 Trans-Blot Cell Lid With Power Cables 170-3943 Trans-Blot Platinum Anode Plate Electrode 170-3944 Trans-Blot Stainless-Steel Cathode Plate Electrode 170-3945 Trans-Blot Plate Electrode Pair, platinum anode and stainless-steel cathode 16 规格:说明: Trans-Blot Plus

电泳转印槽组件 1。缓冲液槽及带有电缆的盖 2。凝胶支架转印夹 3。纤维衬垫 4。电极丝 5。电极板 6。特级冷却芯 Trans-Blot 转印槽是功能灵活的转印设备,可理想地用于多种转印应用。Trans-Blot 转印槽特点包括:*能进行多胶转印,可容纳3 块PROTEAN I xi 凝胶、6块Criterion 凝胶、12块Mini-PROTEAN 3 或Ready Gel 预制胶*多组参数灵活可设,可调节的电压设置(从30 V 的过夜转印到200 V 的1 小时快速实验)*电极间距设置为8 cm 用于标准印迹杂交,或设置为4cm 用于高强度印迹杂交*可选择板式电极:涂有铂金的钛作为正极,不锈钢为负极,能提供高强度电场和比其它电极更高的电流密度。或选择较经济的铂金电极丝*通过特级冷却芯和水循环仪来调节温度―是天然酶(4°C)或高强度转印的理想选择,随着转印时间增加(多达24 小时),不会引起缓冲液耗竭(在高强度转印中必须使用冷却芯,也推荐用于所有板式电极的应用)*带铰链的凝胶支架转印夹能避免滑动,确保凝胶与印迹膜间的紧密接触;每个转印夹都有颜色标记以保证在转印槽中的正确定位 Trans-Blot 转印槽的锁闭凝胶支架转印夹系统。转印夹(1)支撑凝胶(2)印迹膜(3)两侧有纤维衬垫和滤纸(4)确保凝胶三明治内的完全接触。凝胶夹垂直插入缓冲液槽中(5)。

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