Nestin增强部分肝切除小鼠模型肝再生增殖能力的研究

目的探讨巢蛋白(nestin)基因过表达对小鼠部分肝切除后肝再生增殖能力的影响及其可能的分子机制。方法应用荧光定量PCR、Western blot和免疫组化的方法检测转基因小鼠肝脏中外源基因nestin的表达情况。使用Brdu标记法比较转基因小鼠和野生型小鼠肝脏在不同状态下(静息状态、肝叶切除后24 h、肝叶切除后72 h)肝细胞增殖能力的差异。结果荧光定量PCR、Western blot和免疫组化证实转基因小鼠肝脏中存在nestin的高表达状态。在静息状态下,野生鼠和nestin过表达的转基因鼠肝脏BrdU标记率几乎为零,两者无明显差异;但在肝大部切除的情况下,nestin过表达的转基因鼠有更多的细胞进入增殖状态,增殖能力较野生鼠明显增强,差别具有统计学意义(24 h:t=2.996,P=0.024;72 h:t=2.915,P=0.027)。结论 nestin过表达对静息状态下的肝脏增殖无影响,但可增加损伤修复情况下肝脏的再生增殖能力。表明nestin过表达对静止期细胞作用不明显,但在一些病理或刺激状态下nestin可以通过促进DNA的合成而加速细胞的分裂增殖。

Inhalation of hydrogen gas reduces liver injury during major hepatotectomy in swine

AIM： To study the effect of H2 gas on liver injury in massive hepatectomy using the Intermittent Pringle maneuver in swine. METHODS： Male Bama pigs （n = 14） treated with ketamine hydrochloride and Sumianxin Ⅱ as induc- tion drugs followed by inhalation anesthesia with 2% isoflurane, underwent 70% hepatotectomy with loss of bleeding less than 50 mL, and with hepatic pedicle occlusion for 20 min, were divided into two groups： Hydrogen-group （n = 7）, the pigs with inhalation of 2% hydrogen by the tracheal intubation during major hepa- totectomy; Contrast-group （n= 7）, underwent 70% hepatotectomy without inhalation of hydrogen. Hemo- dynamic changes and plasma concentrations of alanine aminotransferase （ALT）, aspartate aminotransferase （AST）, hyaluronic acid （HA）, tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6）, and malondialdehyde （MDA） in liver tissue were measured at pre-operation, post-hepatotectomy （PH） 1 h and 3 h. The apoptosis and proliferating cell nuclear antigen （PCNA） expres- sion in liver remnant were evaluated at PH 3 h. Then we compared the two groups by these marks to evalu- ate the effect of the hydrogen in the liver injury during major hepatotectomy with the Pringle Maneuver in the swine. RESULTS： There were no significant differences in body weight, blood loss and removal liver weight be- tween the two groups. There was no significant differ- ence in changes of portal vein pressure between two groups at pre-operation, PH 30 min, but in hydrogen gas treated-group it slightly decrease and lower than its in Contrast-group at PH 3 h, although there were no significant difference （P = 0.655）. ALT and AST in Hydrogen-group was significantly lower comparing to Contrast-group （P = 0.036, P = 0.011, vs P = 0.032, P = 0.013） at PH 1 h and 3 h, although the two groups all increased. The MDA level increased between the two group at PH i h and 3 h. In the hydrogen gas treated- group, the MDA level was not significantly significant at pre-operation and significantly low at PH 1 h and 3 h comparing to Contrast-group （P = 0.0005, P = 0.0004）. In Hydrogen-group, the HA level was also significantly low to Contrast-group （P = 0.0005, P = 0.0005） al- though the two groups all increased at PH 1 h and 3 h. The expression of cluster of differentiation molecule 31 molecules Hydrogen-group was low to Contrast-group. However, PCNA index （%） was not statistically signifi- cant between the two groups （P = 0.802）. Micropho- tometric evaluation of apoptotic index （AI） in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-stained tissue after hepatotectomy for 3h, the AI% level in the hydrogen was significantly low to Contrast-group （P = 0.012）. There were no significant difference between Hydrogen-group and Contrast- group at pre-operation （P = 0.653, P = 0.423）, but after massive hepatotectomy, the TNF-α and IL-6 levels increase, and its in Hydrogen-group was significantly low compared with Contrast-group （P = 0.022, P = 0.013, vs P = 0.016, P= 0.012）, respectively. Hydro- gen-gas inhalation reduce levels of these markers and relieved morphological liver injury and apoptosis.CONCLUSION： H2 gas attenuates markedly ischemia and portal hyperperfusion injury in pigs with massive hepatotectomy, possibly by the reduction of inflamma- tion and oxidative stress, maybe a potential agent for treatment in clinic.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system

AIM： To explore the feasibility of passage of bone- marrow-derived liver stem cells （BDLSCs） in culture systems that contain cholestatic serum.METHODS： Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction （RT-PCR） and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.RESULTS： The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units （H-CFUs） were formed. H-CFUs expressed markers of embryonic hepatocytes （alpha-fetoprotein, albumin and cytokeratin 8/18）, biliary cells （cytokeratin 19）, hepatocyte functional proteins （transthyretin and cytochrome P450-261）, and hepatocyte nuclear factors 1α and -3β）. They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.CONCLUSION： BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Effect of preoperative FOLFOX chemotherapy on CCL20/CCR6 expression in colorectal liver metastases

AIM： To evaluate the influence of preoperative FOLFOX chemotherapy on CCL20/CCR6 expression in liver metastases of stage IV colorectal cancer （CRC） patients.（/7 = 53） and in patients who did not receive FOLFOX chemotherapy prior to liver surgery （n = 29）. RESULTS： Of the 53 patients who received FOLFOX, time to liver surgery was n〈 1 mo in 14 patients, 〈 1 year in 22 patients and 〉 1 year in 17 patients, respectively. In addition, we investigated the proliferation rate of CRC cells in liver metastases in the different patient groups. Both CCL20 and CCR6 mRNA and protein ex- pression levels were significantly increased in patients who received preoperative FOLFOX chemotherapy ~〈 12 mo before liver surgery （P 〈 0.001） in comparison to patients who did not undergo FOLFOX treatment. Further, proliferation of CRLM cells as measured by Ki-67 was increased in patients who underwent FOLFOX treat- ment. CCL20 and CCR6 expression levels were significantly increased in CRLM patients who had undergone preoperative FOLFOX chemotherapy. CONCLUSION： This chemokine/receptor up-regulation could lead to increased proliferation/migration through an autocrine mechanism which might be used by surviving metastatic cells to escape cell death caused by FOLFOX.

Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells

AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg): LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP=1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (l:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer.

瑞舒伐他汀经PPARγ-LXRα信号通路抑制THP-1巨噬细胞脂质蓄积的实验研究

目的探讨瑞舒伐他汀经过氧化物酶体增殖物激活受体γ-肝脏X受体α(PPARγ-LXRα)信号通路抑制THP-1巨噬细胞脂质蓄积的机制。方法2018年12月—2019年3月于海口市人民医院/中南大学湘雅医学院附属海口医院中心实验室进行实验。设立THP-1巨噬细胞对照组(0 mmol/L ox-LDL)、ox-LDL组(100 mmol/L ox-LDL)及瑞舒伐他汀低、中、高剂量组(100 mmol/L ox-LDL+10.0μg/ml瑞舒伐他汀、100 mmol/L ox-LDL+100.0μg/ml瑞舒伐他汀、100 mmol/L ox-LDL+1000.0μg/ml瑞舒伐他汀),各组细胞置于CO2培养箱,培养72 h。培养结束后,MTT法测定THP-1巨噬细胞活力,流式细胞仪测定THP-1巨噬细胞凋亡水平,贝克曼AU-480型生化仪测定THP-1巨噬细胞TC、FC、CE水平,RT-PCR法及Western-blot法测定THP-1巨噬细胞PPARγ、LXRα基因和蛋白水平。结果与对照组比较,ox-LDL组巨噬细胞OD值、存活率、TC、FC、CE水平明显升高(P<0.05),凋亡率、PPARγ、LXRαmRNA和蛋白水平明显降低(P<0.05),与ox-LDL组比较,瑞舒伐他汀各剂量组OD值、存活率、TC、FC、CE明显降低(F=13.254、24.145、121.321、259.658、368.487,P均=0.000),凋亡率、PPARγ、LXRαmRNA和蛋白水平明显升高(F=19.632、16.145、18.547、21.214、23.148,P均=0.000),且随着瑞舒伐他汀剂量的增加,瑞舒伐他汀各剂量组OD值、存活率、TC、FC、CE逐渐降低,凋亡率、PPARγ、LXRαmRNA和蛋白水平逐渐升高,差异有统计学意义(P<0.01)。结论瑞舒伐他汀能促进THP-1巨噬细胞胆固醇流出,减少THP-1泡沫细胞中的脂质积累;其机制与瑞舒伐他汀促进PPARγ、LXRαmRNA和蛋白表达,进而激活PPARγ/LXRα途径有关。