骨髓间充质干细胞的分离培养及生物学特性研究

目的：通过体外细胞的培养方法将骨髓间充质干细胞由骨髓血中分离出来并加以纯化，进一步在体外培养条件下研究其增殖及生长特征，从而为探讨后续利用组织工程学方法修复肌肉骨骼系统组织缺损的可能性提供实验基础。方法：从骨髓血中提取间充质干细胞，体外培养扩增，再以相差显微镜、电镜观察并绘制生长曲线。结果：（1）通过离体培养，可以使体内环境下低丰度的骨髓间充质干细胞实现数目扩增。（2）体外培养条件下骨髓间充质干细胞具有成纤维细胞的生长特性。（3）体外单层培养条件下贴壁生长的骨髓间充质干细胞会出现“返祖现象”。（4）骨髓间充质干细胞内细胞器与其生物活性变化相一致。结论：骨髓间充质干细胞体外培养成功为今后利用自体间充质干细胞通过组织工程学方法修复肌肉骨骼系统的组织缺损提供功能细胞奠定了基础。     

利多卡因对N-甲基-D-天冬氨酸抑制大鼠肾上腺嗜铬细胞瘤细胞增殖的影响

目的 观察利多卡因对 N-甲基-D-天冬氨酸(NMDA)抑制大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞)增殖的影响。方法 将体外培养的 PC12细胞分为6组，分别采用正常不含药液的培养基(C组)；含400μmol·L(-1)NMDA 的培养基(N组)；NMDA 分别混合10μmol·L~(-1)(L_1组)、10~2μmol·L~(-1)(L_2组)、10~3μmol·L~(-1)(L_3组)以及10~4μmol·L~(-1)(L_4组)利多卡因的培养基培养5d，应用流式细胞仪测定细胞 DNA 相对含量，解析细胞周期，计算 S 期细胞荧光强度占受测细胞总荧光强度的百分数为 S期分数(SPF)和 S 期与 G_2期细胞荧光强度之和与 M 期细胞荧光强度的比值[(S G_2)/M]。结果 与C 组比较，N、L_1组 SPF 和(S G_2)/M 均降低(P<0.05)，L_4组 SPF 降低(P<0.05)，而 L_2及 L_3组 SPF和(S G_2)/M 差异无统计学意义(P>0.05)。与 N 组比较，L_2、L_3及 L_4组 SPF 和(S G_2)/M 升高(P<0.05)，L_1组 SPF 升高(P<0.05)，而(S G_2)/M 差异无统计学意义(P>0.05)。结论 NMDA 可以通过抑制 PC12细胞 DNA 合成而影响细胞的增殖活性，利多卡因能拮抗 NMDA 对 PC12细胞增殖的抑制作用。     

哌嗪雌酚酮对新生大鼠头盖骨成骨细胞的影响

目的 了解新合成化合物——哌嗪雌酚酮对体外培养成骨细胞的影响。方法 应用MTT法、对硝基苯磷酸盐法、von Kossa染色法、流式细胞术及RT-PCR观察哌嗪雌酚酮对体外培养成骨细胞的增殖、分化、矿化结节形成、细胞周期及I型胶原蛋白表达的影响。结果 哌嗪雌酚酮可刺激成骨细胞增殖,刺激骨结节形成(10-7mol/L组,P<0.05)。结论 哌嗪雌酚酮具有刺激体外培养成骨细胞增殖和晚期分化成熟的功能。     

低浓度5-Fu24-小时持续化疗对BEL-7402肝癌细胞株的细胞周期的影响

目的研究低浓度5-Fu 24-小时持续化疗和高浓度5-Fu短时间化疗对BEL-7402肝癌细胞株的细胞周期的影响：方法用低浓度(1000．0μg/L)的5．Fu对BEL-7402肝癌细胞株进行持续24小时的培养(A组)，用高浓度(50000．0μg/L)的5-Fu对BEL-1 7402肝癌细胞株进行2小时培养(B组)，在培养后的不同时间点用流式细胞技术检测细胞周期的变化。结果A组结果显示：0h、4h、8h、12h、16h、20h和24h的S期细胞的百分数分别为25.23％、32．35％、39．28％、41．05％、46．02％、47．00％及47．14％。B组结果显示：0h、4h、8h、12h、16h、20h和24h的S期细胞的百分数分别为24．68％、68．43％、46．67％、43．67％、35．42％、33．22％及32．96％。结论5-Fu引起的S期细胞周期阻滞不但和浓度相关，也和作用时间相关。低浓度(1000．0μg/L)的5-Fu持续化疗较高浓度(50000．0μg/L)的5-Fu短时间(2小时)化疗更容易引起BEL-7402肝癌细胞株S期阻滞。     

Opioid peptides and receptors in joint tissues: study in the rat.

Using immunohistochemical and biochemical techniques, the occurrence of endogenous opioid peptides and their receptors in normal rat bone and joint tissues was investigated. Opioid receptors were detected, quantified, and characterized in homogenates from capsule/synovium and periosteum using radioligand binding assays. Receptor binding of the nonselective opioid [3H]naloxone to tissue homogenates was stereospecific and saturable, showing similar characteristics to that of brain tissue, although with lower binding capacities. By immunohistochemistry, the neuronal occurrence of four different enkephalins was demonstrated in synovium, bone marrow, periosteum, and juxta-articular bone, whereas no neuronal dynorphin immunoreactivity was detected. Double-staining studies disclosed that enkephalins coexisted with substance P in primary afferent fibers. The applied techniques can be used to assess changes in the distribution of endogenous opioids and their receptors in joint tissues in conditions associated with pain and inflammation. The endogenous opioid system now demonstrated might be targeted and exploited therapeutically to obtain peripheral control of symptoms in joint disorders.     

幽门螺杆菌感染对胃黏膜上皮细胞端粒酶活性的影响及其与细胞凋亡的关系

目的探讨幽门螺杆菌(Hp)感染对端粒酶活性的影响及其与细胞凋亡的关系。方法慢性胃炎伴萎缩者16例,伴肠化生者15例,伴中、重度不典型增生者14例,胃癌19例共64例为研究对象。采用快速尿素酶试验、Warthin-Starry银染色检测幽门螺杆菌,采用S-P法检测人端粒酶催化亚单位(hTERT)蛋白表达,采用TUNEL染色法原位检测细胞凋亡。结果(1)萎缩性胃炎、肠化生、不典型增生和胃癌组织中hTERT蛋白表达率分别为25.00%、46.67%、64.29%和78.95%,呈递增趋势。Hp阳性患者hTERT蛋白表达率为77.42%,显著高于Hp阴性患者(33.33%,P<0.01)。(2)Hp阳性伴hTERT蛋白阳性患者细胞凋亡指数为9.57±5.01,显著高于Hp阴性伴hTERT蛋白阳性患者(6.01±5.31,P<0.05)。结论(1)端粒酶激活可能参与了胃黏膜上皮细胞恶性转化的全过程。Hp感染与端粒酶活性密切相关。Hp感染可能是激活端粒酶的重要机制之一。(2)Hp感染既可激活端粒酶,引发细胞永生化,又可以诱导胃黏膜上皮细胞凋亡,进而增加了胃黏膜的不稳定性和癌变的危险性,这可能是Hp致癌的主要机制之一。     

Enzyme-linked Immunosorbent Assay of Pro-gastrin-releasing Peptide for Small Cell Lung Cancer Patients in Comparison with Neuron-specific Enolase Measurement

Our previous study demonstrated that pro-gastrin-releasing peptide(31–98), or ProGRP, is a specific tumor marker in patients with small cell lung carcinoma (SCLC). Using a newly developed, highly sensitive enzyme-linked immunosorbent assay (ELISA) for ProGRP, we analyzed 1,446 samples including those obtained from 478 lung cancer patients to evaluate the clinical usefulness of this ELISA. Several properties indicated that ProGRP is a useful tumor marker for SCLC. First, ProGRP was specifically elevated in SCLC patients. In non-SCLC patients and patients with non-tumorous lung diseases, its serum level was very rarely elevated. Secondly, ProGRP was a reliable marker, in terms of the marked elevation of serum ProGRP levels in SCLC patients. Thirdly, serum ProGRP levels were elevated in SCLC patients even at a relatively early stage of this disease. Fourthly, changes in the serum ProGRP level showed an excellent correlation with the therapeutic responses in SCLC patients. Neuron-specific enolase (NSE) is accepted as a tumor marker of SCLC patients. With the aim of comparing ProGRP and NSE as tumor markers for SCLC patients, we measured serum NSE levels in all samples collected in the present study. We found that ProGRP was superior to NSE in terms of sensitivity, specificity and reliability. Therefore, we consider that ProGRP can play a major role as a clinical tumor marker for SCLC patients.     

The lateral ganglionic eminence is the origin of cells committed to striatal phenotypes: neural transplantation and developmental evidence

In order to determine whether the lateral ganglionic eminence (LGE) of the fetal telencephalon is the primary source of striatal precursors in striatal transplants and tissue cultures, cells derived exclusively from the LGE of fetal rat brains were transplanted into the quinolinic-acid-lesioned striatum of adult rats. After 2–3 months they produced grafts that were almost entirely AChE-positive as well as DARPP-32-, TH-, and calbindin-immunoreactive. The grafts were integrated into the host striatum so that host corticofugal fiber tracts interdigitated with graft tissues similar to the way they penetrate the gray matter of the normal striatum. Fast Blue dye injected into the ipsilateral globus pallidus of LGE grafted produced retrogradely labeled neurons within the grafts, but Fluorogold dye injected into the ipsilateral substantia nigra did not. In a separate experiment using DARPP-32-immunohistochemistry as a striatal marker, fetal (E16) and neonatal (P2) rat brains showed DARPP-32 immunoreactivity in the LGE but not in the adjacent medial ganglionic eminence (MGE). In summary, both fetal LGE cells and LGE grafts express specific striatal markers, and LGE grafts integrate into the host striatum and innervate the major striatal efferent target within the host brain. These data suggest that the LGE is the origin of cells committed to striatal phenotypes in the developing brain.     

Synthesis and pharmacological activity of the N-terminal dermorphin tetrapeptide analogs with CH2-NH peptide bond isosteres

The synthesis of pseudotetrapeptides H-Tyr-D-Ala-Phe-NH-(CH₂)₂-NH₂ (1a), H-Tyr-D-Ala-Phe-ψ(CH₂-NH)-Gly-NH₂ (2a), H-Tyr-D-Ala-ψ(CH₂-NH)-Phe-Gly-NH₂ (3a), and H-Tyr-ψ(CH₂-NH)-D-Ala-Phe-Gly-NH₂ (4a), representing the N-terminal tetrapeptide sequence of dermorphin, in which amide bonds are replaced by CH₂-NH bond, is described. N-acetyl-Tyr and desamino-Tyr pseudopeptide analogs (1-4b), (1-3c) are also described. The analogs were assayed in binding studies based on displacement of μ and δ-receptor selective radiolabels from rat brain membrane and in a bioassay using guinea pig ileum (GPI). Pseudopeptides in which the C-terminal (1a) or D-Ala-Phe (3a) amide bond are substituted, exhibit higher μ-affinities and μ-receptor selectivity than the corresponding Phe-Gly or Tyr-D-Ala analogs (2a, 4a). Acetyl-and desamino-Tyr pseudopeptide analogs (1-4b) and (1-3c) did not exhibit μ and δ-opioid receptor affinity at nM concentration. The relevance of the single peptide replacement and of its association to acetylation or amino group elimination of Tyr, is discussed on the basis of a receptor model for μ and δ opioids.