种子细胞双相接种技术促进组织工程骨体外成熟的研究

目的 比较观察双相接种法和静置接种法构建组织工程骨对种子细胞增殖、分化和成骨表达的影响。方法 取健康志愿者骨髓，密度梯度离心法分离骨髓间充质干细胞并进行体外扩增、诱导分化，然后应用双相接种法和静置接种法与脱钙骨基质（DBM）复合构建组织工程骨，体外培养3周，用倒置显微镜、扫描电镜观察细胞密度、形态及基质分泌情况，并在不同时相点测定组织块中DNA含量、碱性磷酸酶（ALP）活性及钙含量变化。结果 倒置显微镜下双相接种法构建的组织块可见大量具有一定折光性的梭形细胞存在于胶原基质中。扫描电镜见双相接种法构建的组织块切面较平坦，孔隙较小，细胞包埋在胶原中，而静置接种法构建的组织块可见细胞、细胞外基质较少。在体外培养过程中，双相接种法组织块中细胞的DNA含量、ALP活性均高于静置接种法，钙含量在培养2周后高于静置接种法。结论 双相接种法是一种高效的组织工程骨构建方法，有利于提高接种效率和促进组织工程骨的体外成熟。     

Permanent prostate brachytherapy for Japanese men: Results from initial 100 patients with prostate cancer

OBJECTIVE: To evaluate the initial results of brachytherapy for prostate cancer with permanent iodine-125 implant in Japan. METHODS: The results obtained with brachytherapy in the initial 100 Japanese patients treated at Nagano Municipal Hospital were reviewed. Patients with a prostate-specific antigen (PSA) level of less than 10 ng/mL and a Gleason's scores of 5, 6, 3 + 4 were classified as having a low risk of recurrence. Patients with a PSA level of 10-20 ng/mL and/or a Gleason's score of 4 + 3 were classified as having an intermediate risk for recurrence. Seventy-eight of the low-risk patients and 19 of the intermediate-risk patients were treated by seed implants alone, or seed implants combined with preceding external radiation, respectively. A total of 53 patients received neoadjuvant hormone therapy. The efficacy and morbidity of brachytherapy were investigated using the serum PSA, International Prostate Symptom Score, quality of life score and uroflowmetry data. RESULTS: The average V100 and D90 obtained by post-implant dosimetry was 94.3 and 113.7%, respectively. Serum PSA decreased gradually after treatment, although it had still not reached a nadir after 1 year. There was little difference of the PSA level between the patients with and without neoadjuvant hormone therapy even at 1 year after seed implantation. There were no PSA biochemical failure or clinical recurrence during the follow-up period. Voiding symptoms worsened until 3 months after treatment, and then gradually improved. Acute urinary retention occurred transiently in one patient (1%). Rectal bleeding and severe diarrhea did not occur. CONCLUSION: Brachytherapy is a feasible and effective option for the treatment of prostate cancer in Japanese men. Brachytherapy may have a different effect in Japanese patients with respect to voiding symptoms. Urinary retention was rare, but voiding symptoms were persistent in Japanese patients. Neoadjuvant hormone therapy deserves investigation to determine whether it can achieve better results, especially in patients with an intermediate risk.     

Validation of silver-stained nucleolar organizer regions for evaluation of invasive character of urinary bladder carcinoma in rats and mice

A series of 8 rat and 16 mouse invasive bladder carcinomas were investigated for the presence of silverstained nucleolar organizer regions (AgNORs) to clarify whether this parameter is applicable to the estimation of their invasive character. With regard to number of AgNORs per cell, neither rat nor mouse carcinomas showed any difference between invasive and noninvasive sites within the same tumor. However, the frequency of cancer cells bearing bizarre dots, irregular in size and shape, was significantly higher at sites of actual invasion. Quantitative data generated using an image analyzer revealed significantly lower values for NOR roundness and significantly larger NOR size in invasive sites than in noninvasive sites in all groups. Double staining for the proliferation marker proliferating cell nuclear antigen (PCNA) and AgNORs was performed on eight rat carcinomas and a close correlation between the two was confirmed. Thus the number of AgNORs in PCNA-positive cells was significantly greater than in PCNA-negative cells. Furthermore, a particularly strong correlation was observed for incidences of PCNA-positive cells and bizarre dots (P<0.01). The quantitative data also demonstrated significant differences in size and shape of dots. It is concluded that AgNORs have diagnostic value for the invasive character of bladder carcinomas.     

一次手术法对自制两段式种植体种植效果的实验研究

本实验用自制纯钛两段式种植体,以一次手术法植入到狗的下颌前磨牙区,观察5个月,结果表明,观察期间内牙龈无炎症,种植体稳固,X线片显示种植体周围无透射区,镜下可见种植体表面与牙龈之间形成上皮附着样结构,与骨组织之间大多呈骨性愈合。提示了两段式种植体有两种设计和手术方式可供选择。骨内部分在开放的环境中也可达到骨性愈合。     

Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas

 Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal components in uterine carcinosarcoma designated as malignant mixed müllerian tumour (MMMT). We examined the biological properties of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken from a patient with uterine MMMT. We also evaluated c-myc and MYCN gene amplification in three RMS cell lines (including FU-MMT-3) derived from three MMMTs by Southern blot analysis. FU-MMT-3 cells were propagated continuously for 57 serial passages over a 2-year period in vitro. FU-MMT-3 was able to produce tumours demonstrating pure RMS in athymic nude mice. Cytogenetically, FU-MMT-3 showed a triploidy pattern, with complex karyotypic abnormalities including trisomy of chromosome 8. All three RMS cell lines, including FU-MMT-3, showed amplification of the c-myc gene (approximately fourfold to eightfold), while no cell lines demonstrated MYCN gene amplification. FU-MMT-3 is considered to provide a useful system for the study of the biological behaviour of RMS in MMMTs. Extra copies of chromosome 8 and c-myc gene amplification may be associated with the rhabdomyoblastic differentiation in MMMT. Received: 7 January 1997 / Accepted: 2 May 1997     

ELISA检测襄虫病人血清循环抗原及其在疗效考核方面的应用

用快速双抗体夹心ＥＬＩＳＡ检测囊虫病人血清中的循环抗原，其阳性率为６７．５％；正常人血有的假阳性率为５．３３％；包虫、肝吸虫和弓形虫病人血清的交叉反应率分别为６．９％、７．５％和１２．５％。结果表明．该法与常规双抗体夹心ＥＬＩＳＡ相比具有较为敏感、快速、稳定和节省血清、试剂用量的优点，但其特异性有待进一步提高。用快速双抗体夹心ＥＬＩＳＡ和间接型ＥＬＩＳＡ检测第１至１０疗程囊虫病人血清中的循环抗原和抗体，表明检测循环抗原考核囊虫病人的治疗效果优于检测抗体     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Actions on αT3-1 Gonadotrophs Show Desensitization

PACAP is a hypothalamic hypophysiotropic factor that acts upon a number of pituitary cells, including gonadotrophs. In the gonadotroph-derived αT3-1 cell line, PACAP acts via PVR1 receptors to stimulate adenylyl cyclase and phosphoinositidase C. PACAP-stimulated cAMP accumulation is inhibited by protein kinase C-activating phorbol esters in these cells and the current work was undertaken primarily to establish whether it is also subject to homologous regulation. In acute experiments, PACAP27-stimulated cAMP accumulation (intracellular plus extracellular) was measured (in the presence of phosphodiesterase inhibitor) both in intact cells and in cell membranes. The peptide increased cAMP accumulation, but initial rates of PACAP27-stimulated cAMP accumulation were reduced to between 10 and 50% within 10 min of stimulation in both cells and membranes. The initial rate of forskolin-stimulated cAMP accumulation was maintained in membranes but not in intact cells (although the deviation from linearity was less pronounced than with PACAP27). Thus, rapid homologous desensitization to PACAP27 occurs in intact αT3-1 cells, but is not entirely receptor specific. Rapid homologous desensitization of PACAP27-stimulated cAMP accumulation also occurred in the presence of a protein kinase C activating phorbol ester, which inhibited cAMP accumulation without altering the kinetics of the PACAP27 effect. Brief pre-treatment (3 min) with PACAP27 also reduced the ability of PACAP27, but not gonadotrophin-releasing hormone, to cause a spike-type elevation of cytosolic Ca²⁺ concentration (a consequence of phosphoinositidase C activation). In chronic desensitization studies, pre-treatment for 6 h with PACAP27 caused a dose-dependent (IC₅₀ approximately 10 nM) reduction of PACAP-stimulated cAMP accumulation and down regulated cell surface PVR1 receptors (to approximately 50%). Thus, it appears that PACAP27-stimulated (PVR-1 receptor mediated) adenylyl cyclase undergoes rapid homologous desensitization in αT3-1 cells, which is paralleled by homologous desensitization of PACAP27-stimulated phosphoinositidase C activity and involves mechanisms distinct from those underlying heterologous desensitization by phorbol esters. Chronic desensitization of PACAP-stimulated cAMP accumulation and down-regulation of cell surface PVR-1 receptors also occurs in these cells although the receptor loss may not entirely explain the observed desensitization.     

Immunoglobulin heavy chain Diversity genes rearrangement pattern indicates that MALT-type gastric lymphoma B cells have undergone an antigen selection process

Gastric MALT lymphoma usually develops from chronic gastritis, the vast majority of which (>90%) is associated with Helicobacter pylori infection. We sequenced the third complementarity determining region (CDR3) of immunoglobulin heavy chain genes in 19 gastric MALT lymphoma clones to determine the pattern of variable (V), diversity (D) and joining (J) gene utilization during immunoglobulin gene rearrangement.
DNA was extracted from paraffin-embedded sections and the rearranged CDR3 regions were amplified using a semi-nested polymerase chain reaction (with primers complementary to the conserved framework-three segment of the variable region [FR3A] and J regions). The DNA used for cloning and sequencing was obtained after purification of monoclonal bands excised from polyacrylamide gels. The N-D-N region specific to each clone was compared with known germline D sequences.
Similarly to that observed in normal and leukaemic B cells, our series of gastric MALT lymphomas showed apparent preferential utilization of genes from the DXP family. In two cases no similarity between the CDR3 nucleotide sequences of the neoplastic clones and the known germline D sequences could be found. In 10/19 analysed alleles the lymphoma B-cell clones appeared to contain two D gene segments (D-D recombination), a rare occurrence in normal individuals but one which has been described as a significant event in the determination of idiotype expression and antigen-binding affinity. Remarkably, despite the use of different D and J segments, the resultant amino acid sequences matched in two patients, suggesting the presence of a common selecting antigen.
The observed pattern of D gene rearrangement suggests that MALT lymphoma B-cell clones have undergone antigen selection, which seems to indicate that the antigen stimulation plays a pivotal role in the development of the lymphoma.     

Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs.