Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6.

Investigation of human UDP-glucuronosyltransferase (UGT) isoforms has been limited by a lack of specific substrate probes. In this study serotonin was evaluated for use as a probe substrate for human UGT1A6 using recombinant human UGTs and tissue microsomes. Of the 10 commercially available recombinant UGT isoforms, only UGT1A6 catalyzed serotonin glucuronidation. Serotonin-UGT activity at 40 mM serotonin concentration varied more than 40-fold among human livers (n = 54), ranging from 0.77 to 32.9 nmol/min/mg of protein with a median activity of 7.1 nmol/min/mg of protein. Serotonin-UGT activity kinetics of representative human liver microsomes (n = 7) and pooled human kidney, intestinal and lung microsomes and recombinant human UGT1A6 typically followed one enzyme Michaelis-Menten kinetics. Serotonin glucuronidation activity in these human liver microsomes had widely varying V(max) values ranging from 0.62 to 51.3 nmol/min/mg of protein but very similar apparent K(m) values ranging from 5.2 to 8.8 mM. Pooled human kidney, intestine, and lung microsomes had V(max) values (mean +/- standard error of the estimates) of 8.8 +/- 0.4, 0.22 +/- 0.00, and 0.03 +/- 0.00 nmol/min/mg of protein (respectively) and apparent K(m) values of 6.5 +/- 0.9, 12.4 +/- 2.0, and 4.9 +/- 3.3 mM (respectively). In comparison, recombinant UGT1A6 had a V(max) of 4.5 +/- 0.1 nmol/min/mg of protein and an apparent K(m) of 5.0 +/- 0.4 mM. A highly significant correlation was found between immunoreactive UGT1A6 protein content and serotonin-UGT activity measured at 4 mM serotonin concentration in human liver microsomes (R(s) = 0.769; P < 0.001) (n = 52). In conclusion, these results indicate that serotonin is a highly selective in vitro probe substrate for human UGT1A6.     

中国汉族人群遗传性聋基因座位STR位点的遗传多态性研究

目的 研究中国汉族人群10个遗传性聋基因座位的短串连重复序列(STR)的遗传多态性。方法 应用PCR方法对10个位点进行扩增，变性聚丙烯酰胺凝胶电泳分离，记录基因型。采用PPAP软件计算正常人各STR位点等位片段频率、基因型频率、预期基因型频率、多态信息量(PIC)和Hardy-Weinberg平衡吻合性检验。结果 中国汉族人群D6S287、D8S1132、D13S1275、D15S130、D1S2726、D4S3038、D2S2380、D22S282、D14S1042、D7S529各位点分别检出10个、9个、8个、7个、7个、7个、6个、6个、6个及5个等位片段，多态性分布均符合Hardy-Weinberg平衡定律。各位点PIC值分别为0．879、0．854、0．864、0．821、0．686、0．794、0．764、0．732、0．773、0．712；杂合度均高于0．7。结论 中国汉族人群10个位点STR均具有较高的杂合度和多态信息量，是较理想的遗传标记。     

Interleukin-12 enhances the sensitivity of human osteosarcoma cells to 4-hydroperoxycyclophosphamide by a mechanism involving the Fas/Fas-ligand pathway.

Cyclophosphamide (CY) and its derivative ifosfamide are alkylating agents used to treat osteosarcoma (OS). The purpose of these studies was to determine whether alkylating agents affect the expression of Fas ligand (FasL) and whether interleukin 12 enhances the sensitivity of human OS cells to alkylating agents. 4-Hydroperoxycyclophosphamide (4-HC), the preactivated CY compound, and 4-hydroperoxydidechlorocloclophosphamide (4-HDC), its nonalkylating analogue, human OS LM6 cells, and a clone of cells derived by transfection with the interleukin 12 gene (LM6-#6) were used for these studies. Incubation of LM6 and LM6-#6 with 10 micro M 4-HC increased the expression of FasL mRNA (2.5- and 3.0-fold, respectively). By contrast, 4-HDC, Adriamycin (ADR), cisplatin (CDP), and methotrexate (MTX) had no effect on FasL mRNA expression. Increased FasL expression after treatment with 4-HC was also demonstrated by immunohistochemistry and flow cytometry. Drug-induced FasL was functional and mediated cell death. We examined the effect of FasL up-regulation by 4-HC on LM6 and LM6-#6 cells. Flow cytometry showed that LM6-#6 cells expressed 2.2-fold more Fas than LM6 cells. Cytotoxicity of 4-HC, 4-HDC, ADR, CDP, and MTX on LM6, LM6-neo, and LM6-#6 were quantified. Colony-forming assay revealed an IC(50) of 2.10 micro M for 4-HC in LM6-neo cells compared with 0.41 micro M in LM6-#6 cells. The IC(50) for 4-HDC, ADR, CDP, and MTX were not significantly different between the two cell lines. We concluded that the increased expression of Fas enhanced LM6-#6 sensitivity to 4-HC. These data indicate that Fas/FasL may be involved in the cytotoxic pathway of CY. Combining biological agents with chemotherapeutic agents that have complementary Fas/FasL pathway actions may offer new therapeutic alternatives.     

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.     

转移性癌细胞体外水解酶表达及影响因素的作用

目的　为阐明树突状细胞肉瘤 (DCS)细胞在体外培养条件下其水解酶表达的类型及一些影响因素的作用。方法　采用水解空斑法检测 10种特异性蛋白酶抑制剂、不同抗体及基质对 DCS细胞的体外蛋白水解作用的影响。结果　抑肽酶 (aprotinin)、乙二胺四乙酸钠 (EDTA- Na2 )、抑胃酶 ((pepstatin)可显著抑制 DCS细胞的蛋白水解作用。抗泛素抗体、抗 DCS抗体对 DCS的蛋白水解作用无明显影响。在纤粘连蛋白 (FN)、层粘连蛋白 (L N)基质上 DCS细胞铺展良好 ,蛋白水解作用不明显。结论　本实验条件下 ,DCS细胞可表达丝氨酸蛋白酶、金属蛋白酶(非基质水解酶 )、天门冬氨酸蛋白酶的活性。抗泛素抗体、抗 DCS抗体对 DCS细胞的蛋白水解作用无明显影响。不同基质上 DCS细胞水解酶表达状态不同     

自适应滤波提取耳蜗微音器电位初探

为解决表面电极引导耳蜗微音器电位的结果中存在刺激伪迹信号的状况，探讨了用自适应滤波处理抵消刺激伪迹信号提取的方法，结果发现：正常耳的ＣＭ较同侧表面电极引导的波形有一定的滞后时间，２ｋＨｚ以上的神经动作电位波形分化明显；３１耳极重度感音神经性聋和１１耳中度或中重度感音神经性聋耳各频率的ＣＭ消失，９耳极重度感音神经性聋和２９中度和中重度感音神经性聋各频率存在较好的ＣＭ波形。提示：不同程度的感音神经性聋     

细胞凋亡过程中c-fos、p53和c-myc基因的表达

本文主要研究细胞凋亡过程中c－fos、p53和c－myc等基因的表达。方法：经8－Br－cAMP诱导人食管癌Eca－109细胞调亡，应用原位来交技术对c－fos、p53和c－myc基因的表达进行检测。结果：经过诱导的试验组和不经诱导的对照组c－fos、p53和c－myc基因的表达差异有显著性（P＜0．001）。结论：提示这些基因在细胞凋亡的发生发展过程中起着重要的作用。     

抗百日咳毒素单克隆抗体的纯化及应用研究

目的纯化抗百日咳毒素(PT)的单克隆抗体(M cAb),并建立特异、准确的PT定量检测方法。方法采用辛酸-硫酸铵沉淀法和A蛋白亲和层析法纯化杂交瘤细胞腹水,并经阻断抑制试验筛选识别不同表位的M cAb,用于建立定量检测PT的ELISA方法。结果经SDS-PAGE分析,纯化M cAb的纯度均在90%以上,选择二株识别不同抗原位点M cAbs,应用于检测PT的双抗夹心ELISA方法,灵敏度为2.14μg/L,批内变异系数5.85%,批间变异系数9.27%,平均回收率为108.12%,应用该法测定了国内几大生产厂家送检的无细胞百日咳疫苗原液中PT含量。结论获得了纯度高的抗PT M cAb,建立了一种特异、准确的定量检测PT的ELISA方法,并用于无细胞百日咳疫苗原液中PT含量的检测,为无细胞百日咳疫苗的质量控制提供了有力的手段。     

临床超声技术在腹部淋巴结病变检查治疗中的应用

目的　研究分析腹部淋巴结主要病变的超声声像图特征及超声引导活检的临床应用价值。方法　对腹部淋巴结病变进行超声检查和超声引导活检。结果　超声检查可准确显示腹部淋巴结病变的数目、大小、形态、部位及其内部结构 ,能提示与周围组织的关系 ,从而作出诊断与鉴别诊断 ;超声引导能成功进行穿刺活检。结论　超声检查和超声引导活检是诊断腹部淋巴结病变的一种简便、快捷 ,准确、实用的检查手段 ,对明确临床诊断 ,指导临床治疗和观察病变进展与临床疗效 ,均具有良好的应用价值。     

Up-regulation of Niacinamide in Intervertebral Disc Aggrecan in vitro

The regulatory effects of niacinamide （Nia） on intervertebral disc （IVD） aggrecan in vitro was investigated. Chiba＇s 10 ng/mL interleukin-1 （IL-1）-induced rabbit IVD degeneration model in vitro was established. 0.5, 0. 25 and 0.05 mg/mL Nia was added to normal and degenerated IVDs for intervention. On the first and second week after intervention, safranin O-fast green staining intensity and glycosaminoglycan （GS） content were measured. The expression of aggrecan core protein was detected by RT-PCR. The results showed： （1） After treatment with 0. 5 mg/mL Nia for one week, the GS content in nucleus pulposus （NP） was increased by 44.80% as compared with control group （P〈0. 01） ; The GS content in IL-1 induction groups was increased with the increase of Nia concentrations： After treatment with 0. 5 mg/mL for one week, the GS content in NP was increased by 68.30% as compared with control group （P〈0. 01）. After two weeks, GS content in NP and fibrous rings was still higher than in control group at the same period （P〈0. 01） and untreated group （P〈0.01）. （2） Safranin O-fast green staining revealed that with the increase of Nia concentrations, staining density in NP and fibrous rings was increased and histological structure damage to IVDs by IL-1β was alleviated. （3） RT-PCR showed that the expression of core protein gene in IL-1β-induced degenerated IVDS was increased with the increase of Nia concentrations. It was concluded that under conditions in vitro, Nia could up-regulate the expression of aggrecan in IVDs and protect IVDs from IL-1β-induced degeneration at least partially, which offers a potential choice for IVD degeneration clinical therapy.