Midkine expression correlating with growth activity and tooth morphogenesis in odontogenic tumors

Midkine (MK; a low molecular weight heparin-binding growth factor) is a multifunctional cytokine. MK plays a role in morphogenesis of many organs including teeth through epithelial-mesenchymal interactions. We immunohistochemically examined MK expression in various human odontogenic tumors. There was no difference in positive rate and intensity of MK between benign odontogenic tumors and their malignant counterparts. Ameloblastoma showed MK localization in the peripheral columnar cells in budding processes from the parenchyma, which frequently expressed proliferating cell nuclear antigen. MK was also preferentially expressed in keratinized cells in acanthomatous ameloblastoma and keratocystic odontogenic tumor. In odontogenic mixed tumors except for odontoma, intense immunoreactivity to MK was found in epithelial follicles, the surrounding odontogenic ectomesenchymal tissue, and the basement membrane between them. Intensity in the odontogenic ectomesenchyme decreased in relation to distance from the epithelial follicles. No expression was found in tumor cells associated with production of dental hard tissues in odontogenic mixed tumors including odontoma. These findings suggested that MK is involved in the reciprocal interaction between odontogenic epithelium and odontogenic ectomesenchymal tissue in areas without dental hard tissue formation in odontogenic mixed tumors. Coexpression of MK and proliferating cell nuclear antigen was also observed in epithelial follicles and highly cellular nodules in the ectomesenchyme of odontogenic mixed tumors. MK is considered to mediate growth activity of odontogenic tumors and cell differentiation of odontogenic mixed tumors through molecular mechanisms similar to those involved in morphogenesis of the tooth.     

中期因子在肺癌中的研究进展

中期因子(MK)作为肝素结合生长因子家族的一员,参与调控细胞增殖、迁移和诱导细胞恶性转化,并在肿瘤的生长和血管生成等方面起着重要作用.MK在肺癌组织中呈过表达,肺癌患者血清MK水平升高且与预后不良相关.因此,MK有望成为一种新的肿瘤标志物.随着RNA干扰及特异性启动子参与基因治疗等技术的发展,MK可能成为肿瘤基因治疗的新靶点.本文就MK与肿瘤特别是肺癌的关系进行综述.     

Midkine is highly expressed in neuroblastoma tissues

Purpose  Neuroblastoma (NBL) is a tumor from neural crest cells, and is the most frequent solid tumor in children. Midkine (MK) is a pleiotropin analogon, which is frequently expressed in neuronal and epithelial tumors and is a marker for a poor clinical outcome. The aims of this study were to assess MK expression in NBL and investigate the correlation with clinical outcome. Methods  Fifty-six specimens of NBL were stained for MK on a tissue microarray by immunohistochemistry (IHC). Fresh frozen tumor tissues were used for RNA isolation, and RT-PCR analysis for MK-mRNA expression was performed. Survival data, risk factors and disease stages were correlated with MK status assessed by IHC and RT-PCR analysis. Results  MK-mRNA expression was found in the majority of the tumor tissues (75%), whereas MK protein could be detected only in 46% of the NBL by IHC. No correlation of MK status with survival, risk factors or disease stage was observed. Conclusion  A majority of NBL express MK-mRNA, whereas not all MK mRNA positive tumors showed also a positive MK IHC staining. The high expression of MK-mRNA expression might present a promising target for new adenovirus-based gene therapeutic approaches for the treatment of NBL.     

In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles

AIM： To synthesize antisense oligonucleotides （ASODNs） of midkine （MK）, package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma （HCC） growth using these nanoparticles. METHODS： HepG2 cell proliferation was analyzed in vitro using the 3-（4,5-dimethythiazol-2-yl）-5- （3-carboxymethoxyphenyl）-2-（4-sulfophenyl）- 2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction （PCR）. RESULTS： The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs （NANO-ASODNs）. Furthermore, the NANOASODNs significantly inhibited the growth of HCC in the mouse model. CONCLUSION： NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.     

Midkine positively regulates the proliferation of human gastric cancer cells

Midkine (MDK), a heparin-binding growth factor, modulates the proliferation and migration of various cells, is often highly expressed in many malignant tumors, and may act as an oncoprotein. We found that MDK is overexpressed in clinical human gastric cancer tissues relative to its expression in adjacent noncancerous tissues. To further investigate the biological activities of MDK in gastric cancer, we introduced the MDK gene into human SGC7901 gastric cancer cells, where it contributed to the proliferation of SGC7901 cells in vitro and in vivo. Conversely, the knockdown of MDK expression by siRNA resulted in significantly reduced proliferation of BGC823 cells. Our study also shows that MDK activates both the Akt and ERK1/2 pathways and upregulates the expression of several cell-cycle-related proteins, including cyclin A, cyclin D1, Cdk2, Cdk4, and Cdk6, which in part explains the contribution of MDK to gastric cancer cell survival and growth. These results demonstrate that MDK contributes to gastric cancer cell proliferation and suggest that it plays an important role in the development of human gastric cancer.     

血清MK和Th17/调节性T细胞对类风湿关节炎患者病情发生发展的影响

目的 观察Th17/调节性T细胞(Treg)免疫失衡在类风湿关节炎(RA)患者发病时的作用,探讨血清中期因子(MK)和Th17/调节性T细胞(Treg)对RA患者病情发生发展的影响.方法 选取2013年3月至2016年1月在攀枝花市第二人民医院接受治疗的RA患者58例,根据RA是否处于活动期,将26例DAS28≤3.2者纳入非活动组,32例DAS28>3.2纳入活动组,另将30例健康者作为对照组,采用酶联免疫吸附法对三组受检者的血清MK、白介素-17(IL-17)、白介素-6(IL-6)、白介素-23(IL-23)水平进行检测,利用流式细胞术检测调节性T细胞和外周血Th17细胞的比例变化,并对其比例变化和血清MK、IL-17、IL-6、IL-23的水平进行比较.结果 活动组患者的Th17、调节性T细胞比例分别为(1.90±1.00)%、(1.58±1.03)%,非活动组分别为(0.98±0.36)%、(3.92±1.29)%,对照组分别为(0.57±0.21)%、(8.01±1.44)%,活动组血清MK为(342.36±68.39)pg/mL,非活动组为(257.91±56.79)pg/mL,对照组为(200.14±83.42)pg/mL,活动组血清MK及Th17、调节性T细胞比例分别与非活动组比较,差异均具有统计学意义(P<0.05),活动组与非活动组的血清MK及Th17、调节性T细胞比例分别与对照组比较,差异均具有统计学意义(P<0.05).活动组IL-17、IL-6和IL-23分别为(28.74±4.37)ng/L、(12.84±2.38)ng/L、(74.27±11.74)ng/L,非活动组分别为(19.25±3.27)ng/L、(9.36±2.74)ng/L、(52.29±13.83)ng/L,对照组分别为(6.01±3.28)ng/L、(5.99±2.92)ng/L、(27.37±10.27)ng/L,活动组与非活动组的IL-17、IL-6和IL-23水平分别与对照组比较,差异均具有统计学意义(P<0.05).结论 RA患者的血清MK上升,且类风湿性关节炎患者Th17/Treg平衡中Th17相对增高,而Treg会相对降低,MK的上升和Th17/Treg平衡失调都促进了RA的发生发展.     

特异性小干扰RNA下调中期因子基因表达对膀胱癌细胞化疗敏感性的影响

目的 观察中期因子(MK)基因小干扰RNA(siRNA)对膀胱癌细胞化疗药物敏感性的影响.方法 设计合成3个MK siRNA,转染人膀胱癌RT4细胞株,定量RT-PCR方法筛选下调MK mRNA效果最好的siRNA;以该siRNA转染RT4细胞后分组:空白对照组(Con-A)、空载对照组(Con-B)、错配对照组及MK siRNA 3.125、6.25、12.50、25.00、50.00、100.00、200.00 nmol/L组,实时定量RT-PCR和蛋白质印迹方法检测各组细胞MK表达情况;噻唑盐法检测紫杉醇(PTX)对各组细胞生长的影响;通过检测caspase-3活性和TUNEL方法观察癌细胞凋亡情况.结果 3个siRNA均明显下调MK mRNA水平,以S3效果最好.PTX组、Con-A十PTX组、Con-B+PTX组、siRNA 12.50 nmol/L组、3.125 nmol/L+PTX组、siRNA 6.25 nmol/L+PTX组、siRNA 12.50nmol/L+PTX组肿瘤细胞抑制率分别为(18.21±0.36)%、(18.19±0.29)%、(17.89±0.33)%、(1.86±0.52)%、(32.56±0.53)%、(53.83±0.38)%、(78.95±0.55)%.TUNEL结果显示,ConA、Con-B、siRNA 3.125、6.25、12.50 nmol/L组凋亡指数分别为(1.81±0.36)%、(1.89±0.38)%、(5.56±0.58)%、(9.68±0.55)%和(15.25±0.56)%.结果 MK基因siRNA可增强膀胱癌细胞化疗敏感性,诱导凋亡是其重要机制之一.     

中期因子蛋白表达与子宫内膜腺癌进展的关系

目的 探讨中期因子(midkine MK)蛋白在子宫内膜腺癌中的表达和进展的关系.方法 采用免疫组织化学:EnVision法检测72例子宫内膜腺癌组织、15例子宫内膜非典型增生和20例正常子宫内膜组织中MK蛋白的表达情况,并结合患者临床及病理资料进行分析.结果 子宫内膜腺癌、子宫内膜非典型增生、正常子宫内膜组织中MK蛋白阳性表达率分别为63.9﹪、33.3﹪、15.0﹪,子宫内膜腺癌中MK蛋白阳性率明显高于子宫内膜非典型增生(P＜0.01)和正常子宫内膜(P＜0.01).随着肌层浸润深度、TNM分期的增加,MK蛋白的阳性率显著上升(P＜0.01、P＜0.05);有淋巴结转移组MK阳性率明显高于无淋巴结转移组(P＜0.05);MK蛋白阳性的患者总的生存率低于阴性者(P＜0.05).结论 MK蛋白在子宫内膜腺癌恶性进程中表达上调,有望成为子宫内膜腺癌恶性度评估和判断预后的有价值指标.     

自体DC/CIK细胞免疫治疗对EGFR基因突变阴性的晚期肺腺癌患者外周血微转移指标的影响

目的探讨自体DC/CIK细胞免疫治疗对表皮生长因子受体（EGFR）基因突变阴性的晚期肺腺癌患者外周血微转移指标的影响。方法选择EGFR基因突变阴性的晚期肺腺癌患者120例,按随机数字表法分为治疗组和对照组各60例。治疗组进行DC/CIK细胞免疫治疗,对照组仅进行定期随访。采用RT-PCR法检测两组治疗前、治疗1个月和治疗3个月的外周血微转移指标（CEAmRNA、MKmRNA和LunxmRNA）,同时观察两组患者的肿瘤无进展生存时间（PFS）。结果治疗组治疗1个月和3个月后3种指标的表达量均明显低于对照组,差异均有统计学意义（均P＜0.05）。同时两组在治疗1个月和3个月后3种指标的表达量均较治疗前明显上升,差异均有统计学意义（均P＜0.05）。此外,治疗3个月后,对照组有38例患者CEAmRNA达到了108copies/ml以上,有32例患者MKmRNA达到了109copies/ml以上,有35例患者LunxmRNA达到了109copies/ml。治疗组和对照组肿瘤的中位PFS分别是3.10和2.52个月,差异有统计学意义（P＜0.05）。结论自体DC/CIK细胞免疫治疗在EGFR基因突变阴性的晚期肺腺癌患者的维持治疗中达到了初步的疗效,一定程度上延长了生存时间。     

肝素结合细胞因子影响乳腺癌细胞的增殖、侵袭和上皮细胞间质化的功能研究

目的 探讨肝素结合细胞因子(Midkine)的表达对乳腺癌细胞的增殖和侵袭的作用.方法 采用慢病毒介导的shRNA干扰技术在乳腺癌细胞中沉默Midkine的表达,及利用慢病毒高表达Midkine基因在正常乳腺表皮细胞中.利用蛋白质印迹(Western blot)法检测shRNA干扰或高表达后的正常乳腺表皮细胞和乳腺癌细胞中Mid-kine蛋白的表达情况.利用流式细胞仪技术、Transwell小室实验、及细胞划痕实验,分析Midkine基因高低表达对正常乳腺表皮细胞和乳腺癌细胞生长、增殖、侵袭及转移的影响.结果 首先观察了不同乳腺癌细胞中Midkine的表达情况.其中Midkine只有在BT549、MDA-MB157和MDA-MB436间充质乳腺癌细胞(Mesenchymal cell)中表达,而正常细胞和上皮细胞乳腺癌细胞(Epithelial cell)中不表达.BT549细胞中两种不同shRNA降低Mdikine蛋白表达的效率分别达到100%和90%.HMLE正常乳腺上皮细胞中Midkine高表达效率非常高.结晶紫染色法检测细胞增殖实验结果显示,Midkine-shRNAs转染组BT549细胞的生长速度较阴性对照组降低(P<0.01).流式细胞仪检测结果显示,Midkine-shRNAs转染组BT549细胞的增殖指数为(36.06±2.12)%和(32.06±3.46)%,明显低于对照组53.06±3.68%.同时观察到EMT标识物B连环蛋白(β-catenin)、N钙粘蛋白(N-cadherin)在Midkine-shRNAs转染组BT549细胞中表达上调.Transwell小室实验显示两个Midkine-shRNA转染组BT549细胞迁移(Migration)细胞数为零和(7±4.4)个高/倍视野,它们的侵袭(Invasion)细胞数为(38±7)和(46±8)个/高倍视野,明显低于对照组的迁移(Migra-tion)细胞数(178±14)个/高倍视野,侵袭(Invasion)细胞数(232±35)个/高倍视野.同时也观察到高表达Midkine正常乳腺上皮细胞表现出间质化(EMT)的表型,EMT标识物E钙粘蛋白(E-cadherin)、连环蛋白(β-catenin)在Midkine高表达HMLE细胞中表达下调的现象.细胞划痕实验显示Midkine高表达HMLE细胞组的迁移距离明显高于对照组.结论 在BT549细胞中降低Midkine的表达,可抑制BT549细胞的迁移和侵袭,同时可明显上调对上皮细胞间质化标志蛋白β-catenin和N-cadherin表达,提示在Midkine高表达的肿瘤患者中,通过抑制其蛋白表达可能抑制肿瘤的转移和侵袭.