Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro

Background: We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. Methods: After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group 1 TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Results: Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 ± 3.8% CD4⁺ T cells and 5.3 ± 3.3% CD8⁺ T cells, all four TIL cultures showed 80% CD8⁺ TILs and no CD4⁺ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 × 10⁶) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p<0.01). Conclusions: We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.Presented at the 48th Annual Meeting of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.     

直肠癌远端移形黏膜COX-2及BCL-2蛋白的表达与临床意义

目的探讨直肠癌远端移形黏膜COX-2及BCL-2蛋白的表达情况,判断直肠癌远端移形黏膜是否为癌前病变。方法应用高铁二胺-阿辛蓝染色检测54例直肠癌远端2cm处黏膜．将远端黏膜分为移形黏膜（TM）组及非移形黏膜（NTM）组,通过免疫组织化学染色检测TM中COX-2和BCL-2蛋白的表达．比较TM与NTM、肿瘤组织以及正常黏膜组织（20例直肠良性息肉旁肠黏膜组织）内BCL-2以及COX-2的表达的差异。结果54例直肠癌远端2cm黏膜处组织中有19例存在TM．35例为NTM。COX-2蛋白在肿瘤组织、TM、NTM、正常组织中的阳性率分别为81．5％（44／54）、21．1％（4／19）、17．1％（6／35）、10．0％（2／20）;BCL-2蛋白在上述4种组织中的阳性率分别为77．8％（42／54）、21．1％（4／19）、22．9％（8／35）、5．0％（1／20）。TM内的COX-2及BCL．2蛋白的表达与肿瘤组织相比,差异有统计学意义[（0．737±0．895）比（3．519±1．998）;（0．632±0．955）比（2．833±1．756）,均P〈0．01];与NTM、正常肠黏膜组织相比,差异无统计学意义（均P〉0．05）。结论直肠癌远端TM内的COX-2和BCL-2蛋白的表达无特异性．TM是癌前病变的证据不足。     

Detection and Functional Analysis of Tumor Infiltrating T-Lymphocytes (TIL) in Liver Metastases from Colorectal Cancer

Background  Tumor-infiltrating T lymphocytes (TIL) play an important role in primary colorectal cancer, but their activity in liver metastases has not yet been investigated. The aim of this study was to examine whether tumor-selective infiltration, activation, and cytotoxic activity of TIL can be demonstrated in situ in colorectal liver metastases. Methods  TIL were obtained from liver metastases and corresponding normal liver tissue of 16 patients with colorectal liver metastases. Characterization of TIL in situ was performed by multicolor flowcytometric analysis. Presence of tumor antigen-reactive T cells was evaluated by interferon gamma Elispot analysis. Results  TIL in colorectal liver metastases responding against tumor antigens were present in most patients. Although the proportions of CD3⁺ T cells were comparable in liver metastasis and normal liver tissue, metastases contained significantly enhanced proportions of CD4⁺ cells (49% vs. 22%, P < .001). Among all CD4⁺ T helper cells, the proportion of activated (CD4⁺CD25⁺) effector cells was significantly increased in liver metastases (15.0% vs. 7.8%, P = .003). Metastases showed significantly higher proportions of activated (CD69⁺ [70.1% vs. 49.8%, P = .02] and CD25⁺ [4.1% vs. .6%, P = .06]) and cytotoxically active (CD107a⁺) CD8⁺ TIL (3.2% vs. 1.3%, P = .03). Importantly, the presence of activated T helper cells correlated with the frequencies of cytotoxic T lymphocytes that exerted cytotoxic activity in situ (P = .02). Conclusion  CD4⁺ and CD8⁺ TIL are selectively activated in liver metastases, and cytotoxic T lymphocytes exert tumor-selective cytotoxic activity in situ in the presence of activated T helper cells, suggesting the requirement of in-situ-activated T helper cells for efficient cytotoxic T lymphocytes effector function. P. Wagner and M. Koch contributed equally to this study. An erratum to this article can be found at     

大鼠原位肝移植中血红素加氧酶-1表达对BCL-2和VCAM-1影响

目的 探讨血红素加氧酶-1(HO-1)对大鼠原位肝移植后BCL-2和VCAM-1的影响.方法 建立大鼠原位肝移植排斥模型,将大鼠分为三组:对照组(A组),ZnPP组(B组)和CoPP组(C组).三组分别于3 d和7 d处死,取肝脏标本.荧光实时RT-PCR检测HO-1及VCAM-1的表达,免疫组化方法(SP)检测BCL-2的表达.结果 (1)HO-1在C组表达明显升高,B组不明显,A组介于两组之间.(2)VCAM-1在3 d时C组与A或B组相比有明显统计学意义(P＜0.05),A组与B组相比无统计学意义(P＞0.05).7 d时B组与C组相比有明显统计学意义(P＜0.05),A组与B或C组相比无统计学意义(P＞0.05).(3)BCL-2免疫组化染色阳性面积百分率(-x±S,%)统计学分析显示,3 d和7 d时C组与A或B组相比较有明显的统计学意义(P＜0.05),A组和B组相比无统计学意义(P＞0.05).结论 HO-1过表达上调BCL-2的表达,抑制VCAM-1的表达.     

直肠癌远端移行黏膜COX-2及BCL-2蛋白的表达及意义（英文）

目的：探讨直肠癌远端移行黏膜COX-2及BCL-2蛋白的表达情况,判断直肠癌远端移行黏膜是否为癌前病变。方法：应用高铁二胺-阿辛蓝染色（HID/AB）检测54例直肠癌远端2 cm处黏膜,将远端黏膜分为移行黏膜（transitional mucosa,TM）组及非移行黏膜（non-transitional mucosa,NTM）组,通过免疫组织化学（s-p法）的方法检测两组黏膜组织、肿瘤组织以及20例手术切除的直肠良性息肉旁（cm）肠黏膜组织中COX-2蛋白和BCL-2蛋白的表达情况。比较TM内BCL-2以及COX-2的表达与其余各组的表达的差异。结果：54例直肠癌远端2cm处黏膜有移行黏膜19例,非移行黏膜35例。TM和NTM内均可检测到BCL-2及COX-2蛋白的表达,二者与肿瘤内BCL-2及COX-2蛋白的表达有显著差异（P〈0.001）,与直肠良性息肉旁肠黏膜组织中COX-2蛋白和BCL-2蛋白的表达的无差异（P〉0.05）,移行黏膜内COX-2、BCL-2蛋白的表达与非移行黏膜的表达的差别无统计学意义（P〉0.05）。结论：直肠癌远端TM内癌基因蛋白COX-2和BCL-2蛋白的表达无特异性,移行黏膜并非是癌前病变;直肠癌远端切除2 cm已足够。     

Prognostic significance of tumor angiogenesis,Ki 67, p53 oncoprotein,epidermal growth factor receptor and HER2 receptor protein expression in undifferentiated nasopharyngeal carcinoma--a prospective study

BACKGROUND: This study prospectively examines the prognostic role of p53 oncoprotein (p53), Ki67-antigen (Ki67), tumor angiogenesis (MVD), epidermal growth factor receptor (EGFR), and HER2 receptor protein (HER2) expression in Chinese with undifferentiated nasopharyngeal carcinoma (NPC). METHODS: Seventy-eight Chinese were recruited from October 1995 to July 1997 at the Prince of Wales Hospital, Hong Kong. Pretreatment immunohistochemical preparations of the primary tumor were made, and clinical data were collected prospectively until October 30, 2000. The markers were correlated with overall survival (OS), disease-free survival (DFS), time to progression (TTP), and UICC stage. RESULTS: On univariate analysis, EGFR expression correlated with poorer OS (p =.0001), DFS (p =.01), shorter TTP (p =.0001), and advanced T stage (p =.036). Strong EGFR expression, when compared with weak or moderate, was associated with poorer OS (p =.04) and shorter TTP in a subgroup of patients with UICC stage III-IV disease. HER2 expression was associated with advanced UICC stage (p =.006). The presence of p53 expression correlated with poorer DFS (p =.01) and a trend toward shorter TTP (p =.06). No correlation was found with Ki67-antigen or MVD. On multivariate analysis, only EGFR expression was significantly linked to shorter OS and TTP. CONCLUSIONS: EGFR expression in undifferentiated NPC is associated with a poor clinical outcome. A prognostic role of p53 and HER2 expression is suggestive but not consistently defined in this study. The relatively high prevalence of positive staining for EGFR supports the use of molecular targeted therapy in this disease.     

COX-2及Ki67在乳腺浸润性导管癌组织中的表达及临床意义

目的探讨COX-2和Ki67在乳腺浸润性导管癌组织中的表达情况及其临床意义。方法采用免疫组化SP方法检测82例乳腺浸润性导管癌组织与癌旁正常组织中COX．2和Ki67的表达,并结合临床病理特点进行分析。结果82例乳腺浸润性导管癌组织中COX-2和Ki67的表达分别为71．95％和64．63％,与癌旁正常组织相比均明显增高（均P〈0．001）;COX-2和Ki67的表达与肿瘤TNM分期、淋巴结转移、脉管侵犯及组织学分级呈正相关（均P〈0．05）;Ki67的表达与肿瘤大小呈正相关（P〈0．01）;Spearman等级相关分析法显示两者表达呈正相关（P〈O．05）。结论在乳腺浸润性导管癌组织中COX-2和Ki67的表达均增高,两者与乳腺浸润性导管癌临床病理特征密切相关,对两者进行联合检测可反映乳腺浸润性导管癌的生物学行为。     

白细胞介素(IL)-2与IL-4对小鼠膀胱癌肿瘤浸润性淋巴细胞增殖及细胞毒性的协同调节作用

目的 探讨白细胞介素（ＩＬ）－２与ＩＬ－４对膀胱癌肿瘤浸润性淋巴细胞（ＴＩＬ）体外增殖及细胞毒性免疫调控的协同作用。方法 分离膀胱癌ＴＩＬ,置于含ＩＬ－２和（或）ＩＬ－２的完全培养基因中培养４周,定期计数ＴＩＬ增殖数量。四甲基偶氮唑蓝（ＭＴＴ）比色法检测ＴＩＬ细胞毒性。结果 对比单纯ＩＬ－２的培养条件,ＩＬ－２联合ＩＬ－４后４周时ＴＩＬ扩增数量是前者的１．６５倍（Ｐ〈０．０５）。在交靶比为１０：１时,ＴＩＬ对自体膀胱癌细胞（ＢＴＴ７３９）表现出高水平的杀伤活性（Ｐ〈０．０５）。联合培养的ＴＩＬ抗ＢＴＴ３９或小鼠淋巴瘤瘤株（ＹＡＣ－１）的活性与在单纯ＩＬ－２培养的条件下相比无显著改变（Ｐ均〉０．０５）。结论 ＩＬ－４对ＩＬ－２活化的膀胱癌ＴＩＬ增殖具有较强的正向调节效应,而对ＴＩＬ细胞毒性未见明显影响。     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Validation of a Japanese patient-derived outcome scale for assessing total knee arthroplasty: comparison with Western Ontario and McMaster Universities osteoarthritis index (WOMAC)

 We have developed a Japanese self-administered questionnaire based on an English version of the Western Ontario and McMaster Universities osteoarthritis index (WOMAC) to measure subjective function and pain status of patients who undergo a total knee arthroplasty procedure. Using multiple international cohorts, the performance of the developed Japanese scale was compared to the results of the WOMAC in the United Kingdom, United States, Canada, and Australia. The developed scale showed a comparable level of internal consistency and construct/criterion validity. The responsiveness of the scale was superior to the concurrently measured MOS Short Form 36 Physical Function scale. These results suggest that the developed scale is reliable, valid, and responsive for assessing the effectiveness of total knee arthroplasty in the Japanese context despite the cultural life style differences from Western countries. Received: September 9, 2002 / Accepted: December 10, 2002 RID="*" ID="*" Offprint requests to: H. Hashimoto Acknowledgments. We acknowledge the Kinemax Outcomes Group.     

肿瘤浸润淋巴细胞、重组白细胞介素2治疗对手术后原发性肝癌病人细胞免疫功能的影响

对５例经手术切除的原发性肝癌病人用肿瘤浸润淋巴细胞（ＴＩＬ）和重组白细胞介素２（ｒＩＬ－２）治疗。治疗前１周及治疗后１个月检测细胞免疫功能，治疗后外周血淋巴细胞的ＮＫ活性、白细胞介素２产生及活性均明显升高；植物血凝素（ＰＨＡ）皮试阴性转阳性。提示；ｒＩＬ－２和ＴＩＬ应用可明显提高原发性肝癌病人的细胞免疫功能，对杀灭残留癌细胞，抗肿瘤复发、转移，提高远期疗效有一定作用。可能成为肝癌术后抗肿瘤复发，转移的重要疗法。     

24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)₂D₃ [14]. In the present study, we examined whether the effect of 24,25-(OH)₂D₃ on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10⁻⁹–10⁻⁷ M 24,25-(OH)₂D₃. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)₂D₃-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)₂D₃-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)₂D₃-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)₂D₃ on PKC involved phospholipase A₂ (PA₂). Quinacrine (a PA₂ inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)₂D₃, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA₂ plays a role in the nongenomic regulation of PKC by 24,25-(OH)₂D₃. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)₂D₃ modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)₂D₃ regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation. Received: 11 October 1996 / Accepted: 25 April 1997     

Pathophysiological and clinical aspects of the CO2 pneumoperitoneum (CO2-PP)

Experimental studies demonstrated a severe cardiac load of the CO₂ pneumoperitoneum caused by an accelerated after- and a decreased preload. Patients displaying cardiovascular risks are therefore often rejected from laparoscopic surgery. Hence, the pathophysiological changes and the intraoperative risk of the CO₂ pneumoperitoneum in high-risk cardiopulmonary patients (NYHA II–III, n= 15) undergoing laparoscopic cholecystectomy are described. The changes in cardiac after- and preload seem to be due to the elevated intraabdominal pressure rather than transperitoneally resorbed CO₂ and are reversible by desufflation. In one patient conversion to open operation had to be performed because of a severe drop in cardiac output and right ventricle ejection fraction. Mixed oxygen saturation was predicting intraoperative worsening in this case. The described pathophysiological changes may seem to be well tolerated even in high-risk cardiac patients. Monitoring of hemodynamics should include an arterial catheter line and blood gas analyses. Pharmacologic interventions or pressureless laparoscopic procedures might not be necessary as long as laparoscopic cholecystectomy is performed. Received: 13 December 1996/Accepted: 8 January 1997     

Stimulation of Femoral Trabecular Bone Growth in Ovariectomized Rats by Human Parathyroid Hormone (hPTH)-(1-30)NH2

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH₂, which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH₂ to stimulate bone growth despite its having hPTH-(1-31)NH₂'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH₂'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2–10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10–50 nmol of hPTH-(1-30)NH₂/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH₂/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-31)NH₂, hPTH-(1-34)NH₂ and [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-34)NH₂. These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause. Received: 16 September 1998 / Accepted: 15 December 1998     

补肾中药对肾虚骨质疏松症大鼠红细胞膜PKC、 Ca2+-Mg2+- ATP酶活性影响的实验研究

本实验研究切除卵巢造成肾虚骨质疏松症大鼠模型。通过观测红细胞膜蛋白激酶Ｃ（ＰＫＣ）和Ｃａ２＋－Ｍｇ２＿－ＡＴＰ酶的活性，探讨肾虎骨质疏松症病理机制中肌醇脂质系统的变化，并结合全身和脊柱骨密度（ＢＭＤ）指标观察了补肾中药（密骨灵）的疗效，与正常对照组、模型空白组和阳性药（骨疏康颗粒剂）对照组进行对照，探讨补肾法的防治机理。结果表明；模型空白组大鼠红细胞膜ＰＫＣ和Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶、Ｍｇ２＋－ＡＴＰ酶的活性明显低于正常组（ｐ均＜０．０５）；密骨灵组与骨疏康组大鼠全身、脊柱ＢＭＤ和红细胞膜ＰＫＣ、Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶的活性均明显高于模型空白组（ｐ均＜０．０５），并且与正常组大鼠无明显差别（ｐ均＞０．０５）；密骨灵组大鼠红细胞膜Ｍｇ２＋－ＡＴＰ酶活性高于模型空白组和骨疏康组（ｐ＜０．０５），并且与正常组无显著性差异（ｐ＞０．０５）；密骨灵组大鼠红细胞膜ＰＫＣ活性高于骨疏康组（Ｐ＜０．０５）。结论：肾虎骨质疏松症具有红细胞膜ＰＫＣ和Ｃａ２＋－Ｍｇ２＋－ＡＴＰ酶、Ｍｇ２＋－ＡＴＰ酶活性的改变；密骨灵可以恢复肾虎骨质疏松症大鼠全身骨量，达到防治目的；补肾中药可以恢复红细胞膜ＰＫＣ活性和钙镁泵的活性，是补     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Bone Morphogenetic Protein 2 (BMP-2) Enhances BMP-3, BMP-4, and Bone Cell Differentiation Marker Gene Expression During the Induction of Mineralized Bone Matrix Formation in Culturesof Fetal Rat Calvarial Osteoblasts

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling. Received: 27 November 1995 / Accepted: 19 July 1996     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998