鸡腿蘑菌株筛选及深层发酵培养基的优化

对4个鸡腿蘑菌株进行了发酵筛选,选出生物量较高的菌株农林鸡腿蘑(NL),并通过单因素试验,确定玉米粉、蔗糖为碳源,麸皮为氮源;在此基础上,以筛选的碳源、氮源和无机盐KH2PO4和MgSO4·7H2O为考察因素,以生物量为主要指标利用正交试验优化培养基配方比例,确定鸡腿蘑摇瓶发酵培养基最佳配方为:玉米粉 4 g/dL,蔗糖 2 g/dL,麸皮 4 g/dL,KH2 PO4 0.1g/dL,MgSO4·7H2O 0.1 g/dL.     

营养因子对鸡腿蘑分泌胞外多糖的影响

结合工业发酵的特点 ,研究了营养因子对鸡腿蘑 (毛头鬼伞 )胞外多糖产量的影响 ,筛选到适合其胞外多糖分泌的优化培养基配方 :葡萄糖 2 .0 % ,玉米粉 (6 0目 ) 1.0 % ,麸皮粉 (6 0目 )0 .3% ,VB110mg/L ,KH2 PO4 0 .1% ,MgSO4 ·7H2 O 0 .1% ,在此基础上进行摇瓶发酵曲线的测定 ,确定生产胞外多糖的适宜发酵周期为 4d ,发酵液中产量最高可达 96 .3mg/dL .     

羊肚菌液体深层发酵条件

以羊肚菌为材料，研究了发酵过程中碳源、氮源、无机盐、培养条件对菌丝体生物量、胞外多糖的影响，以及发酵过程中菌丝体生物量、胞外多糖、总糖及还原糖质量浓度、培养基PH值的动态变化，并在此基础上确定了羊肚菌液体深层发酵的最佳条件。结果表明：羊肚菌液体深层发酵的最优培养基配比为：玉米粉4．0g／dL、葡萄糖1．0g／dL、黄豆粉2．0g／dL、酵母粉0．3g／dL、KH2PO4 0．2g／dL、MgSO4 0．1g／dL、CaSO4 0．1g／dL；最优培养条件为：24℃，起始pH5．8，250mL的摇瓶装液量为100mL，接种量10mL，摇瓶转速140r／min，发酵时间为108h。     

Penicillium sp.X-1液态发酵生产生淀粉酶的优化

通过摇瓶发酵,研究了培养基成分对Penicillium sp.X-1液态发酵产生淀粉酶的影响。结果表明：碳源、氮源及MgCl2对产酶有较大的影响,经响应面优化得到的培养基组成为：玉米粉42 g/L,豆饼粉30 g/L,MgCl216 mmol/L,在最优条件下酶活达到239 U/mL,与采用基本培养基的相比,酶活提高了7.5倍.     

谷氨酸脱羧酶发酵工艺的优化

用大肠杆菌AS1.505进行液态发酵生产谷氨酸脱羧酶并优化培养基,考察了碳源、氮源、复合营养物质、起始pH及发酵时间对酶活的影响,确定最佳产酶培养基组成为:葡萄糖1.0 g/dL,蛋白胨3.0 g/dL,氯化钠0.3 g/dL,磷酸氢二钾0.1 g/dL,硫酸镁0.02 g/dL,L-谷氨酸0.01g/dL,玉米浆1.5 g/dL,生物素30 t,g/L,麸皮4 g/dL;pH 6.5.在此基础上,设计发酵条件的优化实验.实验结果表明为:250 mL的三角瓶装液量25 mL,37℃,起始pH 6.5,培养18 h达到产酶高峰,产酶活力可达1 290 U/mL.     

黄伞菌丝深层发酵的培养条件

采用摇瓶培养法对黄伞菌丝深层发酵培养条件进行了研究.通过正交试验初步确定黄伞菌丝深层发酵适宜的培养基组成为:葡萄糖3g/dL,牛肉膏1.5g/dL,K2HPO40.5g/dL,MgSO40.1g/dL.该菌株最适培养条件为:培养温度25℃,起始pH值5.0,接种体积分数15%,发酵周期10d.在优化的试验条件下,进行摇瓶发酵,菌丝干重达11.16g/L.     

放射型根瘤菌WSH2601生产辅酶Q10的摇瓶发酵条件

以放射型根瘤菌 (Rhizobusradiobacterium ,WSH2 6 0 1)作为辅酶Q10 的生产菌株 ,研究了氮源、碳源、接种量、溶氧、初始 pH、发酵温度及添加物等因素对细胞生长与产物辅酶Q10 合成的影响 .结果表明 :玉米浆和酵母膏是较好的氮源 ,葡萄糖与蔗糖是辅酶Q10 发酵的较好的碳源 ,接种量对辅酶Q10 发酵的影响不大 ,为 4 % .适宜的初始 pH值为 7,番茄汁能较好地促进细胞的生长 ;添加玉米浆、L 甲硫氨酸、番茄汁和异戊醇有利于产辅酶Q10 ;溶氧对细胞生长与产物辅酶Q10 合成的影响较显著 .通过正交试验初步确定了发酵条件 :碳源为 1.5 g/dL葡萄糖和 2 .5 g/dL蔗糖混合物 ,酵母膏 0 .8g/dL ,初始pH 7,每 5 0 0mL装液量为 5 0mL .最后经综合优化条件 ,在摇瓶发酵条件下 :菌体生长量 (以干重计 )为 13.8g/L ,发酵液中辅酶Q10 产量达到 2 2 .7mg/L ,比优化前分别提高 34%和 5 3% .     

圆弧青霉PG37碱性脂肪酶的发酵工艺条件

研究了圆弧青霉PG3 7碱性脂肪酶的发酵工艺条件 ,优化了PG3 7的摇瓶最适产酶条件 .其中 ,发酵培养基的组成为 (g/dL) :豆饼粉 3 .0 ,玉米浆 3 .0 ,磷酸氢二钾 1 .0 ,硫酸镁 0 .1 ,大豆磷脂0 .5,柠檬酸钠 0 .0 5,花生油 0 .2 ;发酵培养基起始 pH 7.5,发酵温度 (2 9± 1 )℃ ,摇床转速 2 50r/min ,发酵周期 96h ,发酵期间于 3 6,54,72h分别流加 0 .4 g/dL花生油 .在此条件下 ,PG3 7的脂肪酶产率为 2 0 60 μmol/ (min·mL) .PG3 7在 2 5L的实验室小罐中的产酶水平为 1 90 0 μmol/ (min·mL) .     

碳源对绿色木霉ZC产中性纤维素酶的影响

筛选得到一株高产中性纤维素酶的绿色木霉ZC,对其培养基中的碳源进行了优化,探讨了碳源的种类、混合碳源以及碳源与麸皮的比例对其产酶的影响,确定了以4 g/dL玉米秸秆粉、1g/dL麸皮为主的发酵培养基,此培养基中纤维素酶滤纸酶活可达321.12 U/mL.     

新疆苏云金杆菌35高效菌株液体深层发酵培养基的筛选

以对棉铃虫高毒力的苏云金芽孢杆菌35作为研究菌株,采用正交试验法及生物测定验证法,对该菌株发酵培养基进行了优化实验。获得了优化培养基(棉籽饼3.25g/dL、玉米粉1.4g/dL、麸皮1.2g/dL、KH2PO40.11g/dL、FeSO40.0049g/dL)。该培养基与对照培养基相比,发酵液含菌数高达40.8×108cfu/mL,比对照培养基提高了63.7%;发酵液毒力高达66.1%,比对照培养基提高了81.5%。     

OK432-induced killer cell activity: potential method for monitoring immunological complications after renal transplantation.

BACKGROUND: Various clinical and biochemical parameters are currently in use for monitoring allograft rejection. However, the mechanism of allograft rejection is complex and it is frequently difficult to obtain a prompt and accurate diagnosis. We examined the usefulness of OK432-induced killer cell activity as an immunological monitoring system for acute renal rejection after renal transplantation. METHODS: Twenty-four renal transplant recipients, seven patients on haemodialysis, and 10 normal volunteers were enrolled in our study. The killer cell activity of peripheral blood mononuclear cells was induced by culturing these cells with the immunopotentiator, OK432, a heat and penicillin-treated lyophilized powder of the Su-strain of Streptococcus pyogenes. RESULTS: The OK432-induced killer cell activity of renal transplant recipients without acute rejection (stable recipients) was significantly lower than in normal volunteers. In four renal transplant recipients with acute rejection, the killer cell activity was significantly higher than in stable recipients. In three recipients suffering from opportunistic infections, killer cell activity was significantly suppressed compared with stable recipients. CONCLUSIONS: Our new test utilizing OK432-induced killer cell activity is potentially useful for monitoring the immunological state and complications after renal transplantation.     

Effects of estrogen on the proliferation and differentiation of osteogenic cells during the early stage of medullary bone formation in cultured quail bones

Quail bone fragments cultured in the medium containing 17β estradiol (E₂) were examined morphologically. On the endosteal bone surface of bones cultured in serum-containing medium, preosteoblasts were observed and labeled by³H-thymidine after 24 h of culturing. After 48 h of culturing, medullary bones occasionally appeared along the endosteal surface, and their bone surfaces were covered with labeled osteoblasts. These osteoblasts were frequently found when added E₂. On the endosteal bone surface of cultured bones in the serum-free medium alone, bone lining cells were observed and not labeled throughout the culture period. On the endosteal bone surface of bones cultured in the serum-free medium containing E₂, labeled preosteoblasts were seen after 24 and 48 h of culturing. However, osteoblasts did not appear. These findings suggest that estrogen acts directly on the proliferation and differentiation of osteogenic cells.     

A prospective evaluation of blood culture versus standard plate techniques for diagnosing peritonitis in continuous ambulatory peritoneal dialysis

Peritonitis remains a major cause of morbidity in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Culture-negative episodes of peritonitis occur at rates of up to 20%, and in part may reflect inadequate culturing techniques of peritoneal effluent. Through a large, prospective study, the improved sensitivity of a blood culture system, when compared with a standard plate technique (P = 0.001), for the detection of bacterial growth in 67 episodes of CAPD peritonitis is demonstrated. Improved recognition of infections caused by gram-positive organisms, primarily Staphylococcus epidermidis, was especially significant using the blood culture system (P = 0.0001). Because of improved sensitivity and a decreased time to organism identification, particularly with infections caused by S epidermidis, the most common cause of bacterial peritonitis in CAPD patients, we suggest that a blood culture system be the standard means of culturing peritoneal fluid in CAPD patients with peritonitis. The lysis-centrifugation system of culturing peritoneal fluid is also discussed in comparison with the blood culture system.     

Xpert MTB/RIF在脊柱结核诊断及利福平耐药检测中的应用价值

目的:探讨Xpert MTB/RIF技术在脊柱结核诊断及利福平耐药检测中的应用价值。方法:选取109例初步诊断为脊柱结核患者的脓液标本,分别行抗酸染色、BACTEC MGIT 960液体快速培养和Xpert MTB/RIF试验,对比3种检测结核分枝杆菌的敏感性及特异性的差异。对不同方法获取的脓液标本行Xpert MTB/RIF检测,评估脓液标本本身对Xpert MTB/RIF检测结核分枝杆菌效能的影响。以BACTEC MGIT 960液体快速培养药敏试验结果为金标准,分析Xpert MTB/RIF检测利福平耐药的效能。结果:抗酸染色、BACTEC MGIT 960液体快速培养及Xpert MTB/RIF检测的总体敏感性分别为25.92%、48.15%和77.78%。Xpert MTB/RIF检测开放手术、B超定位穿刺和穿刺活检取得脓液标本的敏感性分别为83.78%、76.47%和44.68%。以BACTEC MGIT 960液体快速培养药敏试验结果为金标准,Xpert MTB/RIF检测利福平耐药的敏感性和特异度分别为80%(4/5)和90.70%(39/43)。结论:Xpert MTB/RIF试验对脊柱结核具有较高的诊断价值,同时能对利福平耐药菌株进行检测,脓液标本中结核分枝杆菌含量对Xpert MTB/RIF检测的敏感性影响较大。     

Human Testicular Cell Suspensions in vitro using Post Mortem Material

A method is described for culturing human testicular cells from post mortem material for at least 10 days in a serum free medium. The success of the technique is based on the fact that a layer of testicular cells remains undisturbed on the bottom of a culture flask during culturing and the medium is constantly renewed by a perfusion flow. It is shown that cells form junctions within several hours allowing metabolic coupling, indicating active survival of cells during culture period.     

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW,HTK, and Celsior solutions

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.     

骨髓间充质干细胞的贴壁培养及内毒素对其增殖活性的影响

目的贴壁法分离培养大鼠骨髓间充质干细胞(BMSCs),并观察不同浓度内毒素对其增殖活性的影响。方法采用全骨髓贴壁培养法培养大鼠BMSCs,倒置显微镜下观察细胞形态,免疫细胞化学方法检测细胞CD44、CD29、CD34的表达,流式细胞术检测细胞周期。加不同浓度的内毒素(0、0.01μg/ml、0.1μg/ml、1μg/ml、10μg/ml、100μg/ml)作用24h,用四唑盐比色法(MTT)检测BMSCs的增殖。结果分离培养的细胞三代以后呈均一的成纤维细胞样形态,高表达CD44,但不表达CD34、CD45。80%以上的第三代BMSCs处于G1期。不同浓度的LPS作用24h后各组的平均吸光度值(A)比较有统计学意义(F=3.598,P=0.007);0.01μg/mlLPS组A值与其余各组相比,具有统计学意义(P0.05)。结论全骨髓贴壁培养法可以方便、快捷地获得BMSCs,其在形态学、细胞表面标志物表达和多向分化能力方面具有干细胞生物学特性;0.01μg/mlLPS可明显促进BMSCs的增殖。     

Infection diagnosis after knee-TEP-implantation

AIM: In this study, the accuracy of antigranulocyte scintigraphy as a diagnostic means prior to revision in infected total knee replacement was compared to that of preoperative joint aspiration and laboratory parameters. The most efficient combination of all diagnostic methods was calculated and thus a diagnostic algorithm recommended. The value of PCR was compared to commonly used techniques of microbiological culturing. METHODS: Preoperative diagnostic means for infection of 50 total knee replacements in 45 patients requiring revision surgery, were retrospectively analyzed. Inclusion criteria were the intraoperative microbiological and histological verification of infection. Sensitivity, specificity, negative and positive prediction value of C-reactive protein (CRP) and leukocytes, antigranulocyte scintigraphy with (99m)Tc-labeled antibodies, and preoperative joint aspiration were calculated. Furthermore, the accuracy of the different techniques of culturing was compared to that of the polymerase chain reaction (PCR) based on the intraoperative histological findings. Two blinded examiners evaluated specimens taken intraoperatively according to the criteria of Mirra. RESULTS: We observed a sensitivity of 1.0, a specificity of 0.82, a positive prediction value of 0.83 and a negative prediction value of 1.0 for the antigranulocyte scintigraphy. The sensitivity of preoperative joint aspiration was 0.5, the specificity 1.0, and the positive and negative prediction values were 1.0 and 0.5. Correlated to the intraoperative histological findings the accuracy of PCR and culturing was comparable. The highest accuracy was obtained for blood culture samples. CONCLUSION: Compared to preoperative joint aspiration the antigranulocyte scintigraphy proved to be more sensitive in the diagnosis an infected knee replacement while having a high specificity. An advantage of PCR compared to the common microbiological culturing techniques was not observed.     

A modified culture system for epidermal cells for grafting purposes: an in vitro and in vivo study

A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.     

兔腰椎间盘髓核细胞的培养及形态观察

目的：通过对兔腰椎间盘髓核细胞的培养,观察细胞内的演变,探讨髓核细胞的生物学行为及影响因素。方法：取兔腰椎间盘髓核细胞,在加10％灭活胎牛血清的F12－DMEM液中培养,建立体外髓核细胞培养模型。通过光镜、电镜观察,同时进行细胞活力测定。结果：（1）原代细胞生物学性状最接近体内细胞,传代后细胞呈现衰老现象。（2） 活力测定提示随着培养时间的延长及传代,活力逐渐降低。（3）髓核细胞内细胞器的变化与其生物学活性变化相一致。结论：兔腰椎髓核细胞的体外培养成功为人腰椎髓核细胞的培养奠定了基础。