激光扫描共聚焦显微镜测定细胞内游离钙的方法

本文报道运用激光扫描共聚焦显微镜观察细胞内游离钙的测定技术。用Ｆｌｕｏ－３／ＡＭ进行染色，在３７℃、５％ＣＯ２的孵育箱内孵育６０分钟，用激光扫描共聚焦显微镜进行荧光监测。药物最适应的ｐＨ值为７４。此方法已成功地运用于多种细胞内游离钙的测定研究上，大量实验结果表明，本方法灵敏、简单、快速，能实时监测细胞内游离钙的变化。     

肝癌患者外周血树突状细胞的体外扩增、鉴定及内吞能力观察

目的　从肝癌患者外周血培养扩增树突状细胞，观察其形态、表型、内吞及递呈抗原能力，为树突状细胞在肝癌生物治疗中的应用奠定基础。方法　从肝癌患者外周血中分离单个核细胞，在 Ｇ Ｍ Ｃ Ｓ Ｆ、 Ｉ Ｌ４ 的诱导下培养扩增树突状细胞。光学显微镜下观察其体外培养过程中的形态特征及变化，电镜观察其超微结构， Ｆ Ａ Ｃ Ｓ观察其表型特征， Ｍ Ｌ Ｒ检测其抗原递呈能力， Ｈ Ｒ Ｐ内吞实验检测其群体内吞能力。结果　肝癌患者外周血树突状细胞具典型的形态及表型特征，较巨噬细胞更强的激发同种混合淋巴细胞反应的能力。其群体的内吞能力约在培养第 ５ 天达高峰，之后有明显下降。结论　从肝癌患者外周血中可获取较大量的、高纯度的、具较强抗原递呈能力的树突状细胞，在其具有活跃的内吞能力时，以合适的肿瘤抗原致敏可望成为肝癌生物治疗的崭新手段。     

双甲基氨氯吡咪对人肺癌细胞的酸化作用及其意义

目的　研究Ｎａ＋／Ｈ＋ 交换蛋白１（ＮＨＥ１）在肿瘤细胞ｐＨ 调节和增殖／ 死亡调控中的作用，探索肿瘤治疗新策略。　方法　用荧光探针法检测经双甲基氨氯吡咪（ＤＭＡ） 处理后的ＰＬＡ８０１Ｄ 细胞内ｐＨ（ｐＨｉ），同时用活细胞计数法观察ＤＭＡ 对ＰＬＡ８０１Ｄ 细胞增殖的抑制作用。　结果　５０ μｍｏｌ／Ｌ ＤＮＡ 处理４ ｈ 后即能明显酸化ＰＬＡ８０１Ｄ细胞（ Ｐ＜ ０．０５ ～０．０１） 。ＤＭＡ 有显著抑制ＰＬＡ８０１Ｄ 细胞的增殖，并导致其死亡。　结论　ＮＨＥ１ 在肿瘤细胞ｐＨ 调节中起着重要作用，阻止这一调节能抑制肿瘤细胞的增殖并诱导其凋亡     

bcl—2反义寡核苷酸抑制HL—60细胞增殖的研究

目的 研究ｂｃｌ－２反义寡核苷酸在抑制ＨＬ－６０细胞增殖和下调细胞内ｂｃｌ－２蛋白表达水平的作用。方法 将人工合成的ｂｃｌ－２反义寡核苷酸片段与ＨＬ－６０细胞共培养,在作用的第０天,１天,３天,５天通过细胞计数和锥虫蓝染色法检测细胞的生长,存活情况,同时通过免疫组化法（ＡＰＡＡＰ法）检测细胞内ｂｃｌ－２蛋白表达水平的变化情况,结果 ｂｃｌ－２正,反义寡核革酸均可抑制ＨＬ－７０细胞增但ｂｃｌ－２反义     

结肠癌细胞内吞活动中癌基因产物cortactin 的作用

 目的探讨癌基因EMS1表达产物皮层蛋白(cortactin)与结肠癌细胞内吞运动的关系。方法采用共聚焦显微镜检测人结肠癌细胞株HT29细胞内cortactin与内吞过程功能蛋白Rab5及Rab11的分布情况,以及cortactin和组成性内吞标记转铁蛋白(Tfn)的关系。采用病毒转染的方法,将外源性全长cortactin及其突变体转入结肠癌细胞内,观察过表达皮层蛋白及其变异体对细胞内吞活动的影响。进一步采用Western blot和免疫荧光分析方法检测cortactin磷酸化与Tfn内吞在时间和剂量上的关系。结果cortactin与Rab5/11及Tfn在HT29细胞内共定位。当HT29细胞过表达皮层蛋白氨基端或羧基端缺失的突变体时,细胞的内吞活动受到削弱,而表达全长皮层蛋白或载体的对照组未受明显影响。同时发现cortactin的磷酸化与Tfn内吞呈时间和剂量依赖关系。结论cortactin在结肠癌细胞HT29的内吞活动中发挥作用,其功能的发挥可能依赖其氨基端和羧基端结构区域。在内吞过程中,皮层蛋白发生磷酸化。     

人膀胱癌多药抗药性建立及逆转的实验研究

目的建立抗药细胞模型，研究维拉帕米和奎宁对膀胱癌抗药性的逆转及其机制。方法应用阿霉素（ＡＤＭ）大剂量短暂冲击结合低浓度递增法，建立抗药细胞模型，测定抗癌药物５０％抑制浓度和细胞内阿霉素浓度。结果获得了具有多药抗药性的ＢＩＵ－８７／ＡＤＭ细胞，该细胞对ＡＤＭ的敏感性下降了２１倍，同时对表柔比星（表阿霉素，ｅｐｉｒｕｂｉｃｉｎ）、长春新碱、依托泊甙也具有显著的交叉耐药性，对顺铂、丝裂霉素、氟尿嘧啶和甲氨蝶呤无抗药性。ＢＩＵ－８７／ＡＤＭ细胞在脱离ＡＤＭ诱导第８周后，其抗药性保持９０％以上。ＢＩＵ－８７／ＡＤＭ细胞内ＡＤＭ聚积量显著低于ＢＩＵ－８７细胞（Ｐ＜０．０５）；维拉帕米、奎宁能使ＢＩＵ－８７／ＡＤＭ细胞内ＡＤＭ聚积量增加（Ｐ＜０．０５），两者联合应用基本上可逆转ＢＩＵ－８７／ＡＤＭ细胞的抗药性。结论（１）ＢＩＵ－８７／ＡＤＭ细胞具有多药抗药性（ＭＤＲ）表型，且抗药性稳定。（２）细胞内ＡＤＭ聚积量减少是抗药细胞的重要变化，维拉帕米、奎宁能逆转ＢＩＵ－８７／ＡＤＭ细胞的抗药性，可能与增加细胞内ＡＤＭ聚积量有关。     

热休克对不同细胞RNA和HSP 70mRNA合成功能的影响

４５℃加热１０分钟可以杀死５０％的中国仓鼠卵巢（ＣＨＯ）细胞和５７％的Ｈｅｌａ细胞，热休克强烈地抑制ＲＮＡ的合成，使细胞内ＲＮＡ水平急剧降低，Ｎｏｒｔｈｅｍｂｌｏｔ对ＨＳＰ７０ｍＲＮＡ的分析表明热休克并不影响细胞内已有的少量ＨＳＰ７０ｍＲＮＡ但热休克ＨＳＰ７０ｍＲＮＡ的合成会迅速增加。这将改变细胞的生长调控和促进受损细胞的快速增长。     

CpG-ODN对树突状细胞的分化成熟的影响

目的：探讨ＣｐＧ－ＯＤＮ对小鼠骨髓来源的树突状细胞（ｂｏｎｅ ｍａｒｒｏｗ－ｄｅｒｉｖｅｄ ｄｅｎｄｒｉｔｉｃ ｃｅｌｌｓ,ＢＭＤＣ）分化成熟的影响。方法：利用合成的含非甲基化ＣｐＧ基序的寡核苷酸（ＧｐＧ ｍｏｔｉｆ ｃｏｎｔａｉｎｉｎｇｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ,ＣｐＧ－ＯＤＮ）,通过酶切鉴定ＤＮＡ制品ＣｐＧ基序甲基化程序,ＦＡＣＳ分析ＤＣ表型和内吞作用的变化,ＥＬＩＳＡ检测ＤＣ培养上清     

口服／静注葡萄糖及皮下注射胰岛素后肿瘤内pH的变化

快速降低肿瘤内ｐＨ可增加肿瘤细胞对热疗的敏感性。测试了５８例表浅肿瘤内ｐＨ值，其中５０例（８６％）的基础ｐＨ值低于正常组织，中位７．２。肿瘤内各点处的ｐＨ也不尽相同，有１１例肿瘤测２～３个点的ｐＨ值，其值相差在０．０３～０．６０，中位数０．１８。２５例患者口服葡萄糖１００ｇ，有６０％的肿瘤内ｐＨ值下降０．１６±０．０３。１８例口服葡萄糖２００克可使肿瘤内ｐＨ值下降比例从６０％增至８３％，但下降幅度变化不大。静注葡萄糖５０ｇ可达到口服葡萄糖２００ｇ的效果；静注葡萄糖５０ｇ合并口服葡萄糖１００ｇ不能使更多的肿瘤内ｐＨ降低，但可使ｐＨ下降的时间从１２０分钟提前到６０分钟。口服葡萄糖合并皮下注射４０单位胰岛素不能改变肿瘤内ｐＨ下降比例和下降幅度。试验结果证实口服／静注葡萄糖可使肿瘤内ｐＨ快速暂时下降。     

环六亚甲基二乙酰胺对G1期人胃癌MGc—803细胞理化的影响

环六亚甲基二乙酰胺（ＨＭＢＡ）作为一种诱导分化剂，最早用于小鼠红白血病细胞（ＭＥＬＣ）的诱导分化。用ＨＭＢＡ研究人胃癌细胞的诱导分化效应，将有助于了解各种癌症及白血病的发病机理。以人胃癌Ｇ１期ＭＧｃ－８０３细胞经５ｍｍＨＭＢＡ培养液处理后，细胞内环腺苷酸（ｃＡｍｐ）及蛋白激酶Ａ（ＤＫＡ）活性上升，而二酰基甘油（ＤＧ）水平有蛋白激酶Ｃ（ＰＫＣ）活性下降，提示ＨＭＢＡ对细胞诱导分化作用与细胞内信号传导     

The sensitivity of a malignant cell line to hyperthermia (42 degrees C) at low intracellular pH.

The postulate that low intracellular pH acts as a preconditioner for the destructuve effects of hyperthermia (42 degrees C) was examined, using a heat-sensitive line of malignant cells derived from rat mammary gland (SDB). Intracellular pH (pHi) was measured indirectly, from the distribution of the weak, non-metabolizable organic acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) between intra- and extra-cellular water. Respiration, aerobic and anaerobic and anaerobic glycolysis of the cells were studied at normal pHi (pH 7-0-7-4) or at low pHi (pH 6-2-6-6) and at 38 degrees C or 42 degrees C over 6 h in Warburg manometers; the ability of the cells to replicate in culture was examined after 3 h or 6 h incubation in the flasks. The relationship between pHi and extracellular pH (pHe) depended upon the buffer system used and the exact pH in question; no assumption regarding pHi based only on pHe measurement could be made. At 38 degrees C and low pHi, the Pasteur effect became negative due to a relatively greater inhibition of anaerobic than aerobic glycolysis. Respiration was unaffected and cell replicative ability unimpaired. At 42 degrees C and normal pHi, respiration was totally inhibited after 4 h and the Pasteur effect was decreased, in this case due to a compensatory increase in aerobic glycolysis without alteration in anaerobic CO2 production. Low pHi in the presence of hyperthermia enabled cell respiration to continue at a reduced level with no further change in glycolysis. There was delayed cell replication after 3 h at 42 degrees C and inability to multiply following 6 h hyperthermia: low pHi did not influence these results. It is concluded that with these cancer cells, pHi values maintained in the region of 1-0 pH unit below normal for 6 h had no deleterious effect on the cells. No sensitizing effect of the low pHi for the destructive effect of hyperthermia on the cells was observed.     

Regulation of intracellular pH in tumor cell lines: influence of microenvironmental conditions.

The effect of microenvironmental factors on the regulation of intracellular pH (pHi) in MGH U1 cells and EMT-6 cells was studied using the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange were found to be present in both cell types. The activity of both exchangers was dependent on pHi, with low levels of activity at neutral pH and an increase in activity as pHi fell. The level of extracellular pH (pHe) also influenced the operation of the exchangers, with a fall in activity as pHe was reduced over the range 7.4-6.6. This effect was more marked for the Na(+)-dependent Cl-/HCO3- exchanger than for the Na+/H+ antiporter, suggesting that under conditions of reduced pHe the Na+/H+ antiporter is the major mechanism for regulation of pHi. Neither 6 h of radiobiological hypoxia nor variations in the extracellular [Ca2+] over the range 1-6 mM had an effect on the regulation of pHi, while extracellular lactate (5-10 mM) caused a small, concentration-dependent decrease in the combined activity of both exchangers. We conclude that under the microenvironmental conditions found in some regions of tumors, Na+/H+ exchange may be the major method of regulation of pHi.     

血清miR-122和miR-570联合检测对原发性肝癌的临床诊断价值

目的:检测原发性肝癌(PHC)患者血清中miR-122和miR-570的表达水平及临床应用价值。方法:选择2017年2月至2019年1月我院收治的30例原发性肝癌患者作为研究组,选择我院同期30例健康体检者为对照组。采用实时荧光定量PCR方法检测血清miR-122和miR-570的表达水平,利用受试者工作特征曲线评估血清miR-122和miR-570的诊断效能。结果:miR-122和miR-570在原发性肝癌患者血清的表达水平显著低于健康对照者的表达水平,差异具有统计学意义（均P<0.01）;miR-122表达水平与原发性肝癌TNM分期、病理分级、原发性肝癌远处转移程度呈负相关（P<0.05）,miR-570表达水平与原发性肝癌TNM分期、病理分级、原发性肝癌远处转移程度无相关性;miR-122诊断原发性肝癌的AUC为0.816,敏感度为80.5%,特异度为 90.1%;miR-570诊断原发性肝癌的AUC为0.862,敏感度为83.3%,特异性为90.0%;联合血清miR-122和miR-570诊断原发性肝癌的AUC为0.903,敏感性为90.0%,特异性为83.6%。结论:原发性肝癌患者血清miR-122和miR-570降低,血清miR-122表达水平与原发性肝癌TNM分期、病理分级、原发性肝癌远处转移程度呈负相关,联合检测血清miR-122和miR-570对于原发性肝癌的检出具有较高的参考价值。     

Effects of acute pH 6.6 and 42.0 degrees C heating on the intracellular pH of Chinese hamster ovary cells

Incubation of Chinese hamster ovary cells in pH 6.6 medium for 4 h prior to and during 42.0 degrees C heating enhanced thermal cell killing compared to cells heated under normal pH 7.3 conditions. We examined the relationship between the extracellular pH and intracellular pH (pHi) of Chinese hamster ovary cells using a flow cytometer with the pH-sensitive fluorescent molecule 2,3-dicyanohydroquinone. Using either normal (7.3) or low (6.6) pH conditions, the mean pHi and population pHi heterogeneity was studied as a function of time at 42.0 degrees C. Cells incubated at pH 6.6 for 4 h had a resting pHi 0.14 to 0.19 pH units lower than cells at normal pH 7.3, indicating the presence of an active pHi regulatory system. Heating 1 h at 42.0 degrees C at normal pH caused an increase in the pHi of 0.14 pH units. With further heating the cells gradually returned to the unheated (7.3) control levels. Similar pHi changes were observed with the cells incubated and heated at pH 6.6. However, the mean pHi was always more acidic than cells heated at normal pH. Active pHi regulation was still possible for a substantial (greater than 30%) number of cells even after 10 h of heating under low pH conditions. These results suggest that a breakdown in pHi regulation is not the mechanism of low pH-induced heat sensitization.     

Acute extracellular acidification reduces intracellular pH, 42 degrees C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.     

Reduction of intracellular pH as a possible mechanism for killing cells in acidic regions of solid tumors: effects of carbonylcyanide-3-chlorophenylhydrazone

The environment of cells within solid tumors is known to be acidic relative to that in normal tissue, and the viability of tumor cells may depend on mechanisms which maintain intracellular pH (pHi) above the extracellular pH (pHe). We have assessed therefore the toxicity in vitro of the proton ionophore carbonylcyanide-3-chlorophenylhydrazone (CCCP), since this agent has been reported to be capable of transporting H+ equivalent through artificial lipid bilayers and mitochondrial membranes. CCCP was toxic to the human bladder carcinoma cell line MGHU1 and to the murine mammary sarcoma cell line EMT-6 only at pH, less than 6.5. CCCP transported H+ equivalents through cell membranes at physiological (7.35) and low pHc (6.20). Cell lines were found to have steady-state pHi values approximately 0.1 to 0.2 pH units above pHc at pHc less than 6.50. Addition of CCCP led to a decrease in steady-state pHi values as compared to untreated cells at pHc less than 6.50, whereas there was no apparent effect of CCCP on steady-state pHi values at pHc greater than 6.50. The CCCP-induced reduction in steady-state pHi combined with the uncoupling of oxidative phosphorylation by CCCP appeared to be the major mechanisms leading to cell death at pHc less than 6.50. The toxicity of CCCP under acidic conditions was enhanced by amiloride and 4,4'-diisothiocyanostilbene-2,2-disulfonic acid, agents which are known to inhibit membrane-based ion exchange mechanisms which regulate pHi under acidic conditions. When both agents were combined with CCCP, cell killing was observed at pHc less than 7.30. Our results suggest that mechanisms which regulate pHi under acidic conditions which occur in solid tumors may represent targets for new forms of tumor-specific therapy.     

Suppression of the proliferation of cancer cell lines, KB-3-1 and K562 cells preceded by a decrease in intracellular pH caused by phenoxazine derivatives

The intracellular pH (pHi) of cancer cells such as the KB-3-1 (human epidermoid carcinoma cell line) and K562 cells (human chronic myeloid leukemia cell line) cultured in a medium (pH 7.4) was found to be much higher (pH 7.65 and 7.8, respectively) than that of normal cells (pHi is usually < or =7.2). When a phenoxazine derivative, 2-aminophenoxazine-3-one (Phx-3) or 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) was added to these cells, pHi rapidly decreased within 20 min, dose-dependently, though the extent of the decrease of pHi was significantly larger for Phx-3 (a decrease of 0.9 units) than for Phx-1 (a decrease of 0.4 units). Phx-3 and Phx-1 caused the proliferative suppression of these cells 24 h after the addition, dose-dependently. The anti-proliferative effects of Phx-3 on KB-3-1 and K562 cells were far greater than those of Phx-1. It was proposed that the proliferative suppression of KB-3-1 and K562 cells caused by Phx-3 and Phx-1, may be preceded by a rapid and extensive decrease in pHi, which possibly influenced the intracellular homeostasis, finally causing the suppressed proliferation and apoptosis of these cancer cells. The present results suggest that the anti-cancer effects of Phx-3 and Phx-1 may be strengthened by the intracellular acidification of cancer cells by these compounds.     

Increased cytosolic pH in multidrug-resistant human lung tumor cells: effect of verapamil

In a set of four increasingly multidrug-resistant variants of SW-1573 human lung tumor cells, the pHi (i.e., steady-state cytosolic pH) increased up to 0.44 U as a function of the level of doxorubicin resistance. The elevated pHi in the most resistant (2,000-fold) variant dropped to the control level upon addition of verapamil, a known inhibitor of P-glycoprotein activity. These data suggest that, in the absence of xenobiotic substrates, P-glycoprotein activity can affect cellular pHi. This finding may be important for the elucidation of the physiological function of this protein.     

Intracellular acidification of human melanoma xenografts by the respiratory inhibitor m-iodobenzylguanidine plus hyperglycemia: a 31P magnetic resonance spectroscopy study

In vivo 31P magnetic resonance spectroscopy demonstrates that human melanoma xenografts can be significantly acidified by induction of hyperglycemia combined with administration of m-iodobenzylguanidine (MIBG), an inhibitor of mitochondrial respiration. In melanoma xenografts (< or =8 mm diameter), intracellular pH (pHi, measured by the chemical shift of the Pi resonance) and extracellular pH (pHe, measured with 3-aminopropylphosphonate) was reduced by less than 0.2 unit during i.v. infusion of glucose for 40 min. Administration of MIBG (30 mg/kg) under hyperglycemic conditions (26 mM) reduced tumor pHi and pHe by approximately 0.4 (P < 0.001) and approximately 0.6 (P < 0.001) unit, respectively; coincidentally, the nucleoside triphosphates:Pi ratio decreased approximately 60% (P < 0.004) relative to the baseline level. Minimal changes in pHi and pHe and a small decrease in nucleoside triphosphates:Pi ratio (26%, P = 0.2) were observed in liver in response to MIBG plus hyperglycemia. These results suggest that under normoglycemic and hyperglycemic conditions, small human melanoma xenografts (< or =8 mm) may exhibit a relatively high level of oxidative phosphorylation that may be blocked by MIBG. The acidification may result from increased lactate production as a direct effect of MIBG inhibition of respiration in mitochondria of tumor cells, or through indirect systemic effects, which remain to be identified. The synergetic effects of MIBG and hyperglycemia result in significant acidification of the tumor and a decrease in tumor bioenergetic status, and the effects are largely selective for tumors in comparison with normal tissues.