OBJECTIVE To provide information service support for research on proteolysis targeting chimera （PROTAC） in China and provide reference for technical development and patent layout of relevant drug research and development institutions. METHODS The patent analysis method was used to search the patent applications related to PROTAC technology that had been applied to China National Intellectual Property Administration and had been issued before Feb. 2022， using the HimmPat patent database as the search platform. The patent application trend， technology life cycle， main applicants， technology source countries， technology themes， improvement routes and other patent data were analyzed. RESULTS & CONCLUSIONS A total of 133 patents were included in this study. The patent application for PROTAC technology started relatively late in China， with the number of applicants increasing from 2 in 2015 to 30 in 2020， and the number of applications increasing from 2 in 2015 to 38 in 2020. Both the annual patent application volume and the number of applicants were in a period of rapid growth， but the average annual application volume of a single applicant was still less than 2， indicating that research in this field was still in the early stage of technology development； the number of applications from Arvinas， Hisco， and Hinova Pharmaceutical Inc. ranked among the top. Although the number of domestic applications led that of foreign applications in China， the average number of simple peer applications and the average number of simple peer countries in domestic patent applications was only 1.5， which was far lower than that of foreign applications in China， reflecting that there was still room for improvement in the “quality” level of domestic applications. The initial improvements in PROTAC technology mainly focused on the selection of E3 ligands， targets and ligands， and then new improvements such as new PROTAC development， linker design and matching methods emerged， indicating that the patent applicant had started a multi-track layout in the early stages of the development of PROTAC technology. It is suggested that the research and development of PROTAC drugs in China should focus on improving the oral bioavailability and biosafety of PROTAC drugs， overcoming potential drug resistance， and exploring rational design and evaluation methods.

To determine the content of dendrobine in Dendrobium nobile from different harvest times and plant parts, to research the inherent rule about it. GC with internal standard was used to determine. The content of dendrobine had significant differences in different periods and parts. The dendrobine content is higher in four-year root than in three-year root. The dendrobine content in the upper segment of stem is the highest, secondly is in the middle seg-ment, and in the low segment is the lowest. This offered evidence to determine the most appropriate harvest time and fair use of different parts for D.nobil.

Objective To identify clinicopathologic factors which predict survival following hepatectomy in HBV-related cirrhotic patients with early hepatocellular carcinoma (HCC).Methods A database was used to identify patients with histologically confirmed early HCC (≤5 cm,no nodal involvement,metastases,or major vascular invasion) who underwent surgical resection (excluding ablation or transplantation).Among 20 700 patients with HCC who were diagnosed at the Eastern Hepatobiliary Surgery Hospital from April 2005 to November 2010,537 (2.6％) patients with early HCC were studied retrospectively.Prognostic factors were evaluated using the Kaplan-Meier curves,Cox proportional hazards models and the receiver operating characteristic (ROC) curves.Results The study included 537 patients.The median tumor size was 2.9 cm,and 33％ of patients had tumors ≤2 cm.Most HCC lesions were solitary (63％) and had no evidence of vascular invasion (64％).Following surgery,the overall median and 5-year survival were 45 months and 33％ respectively.After adjusting for demographic factors and histological grade,tumor size ＞2 cm (hazard ratio [HR]:1.56),multifocal tumors (HR:1.34),and vascular invasion (HR:2.03) remained independent predictors of poor survival (all P ＜ 0.05).Based on these findings,a prognostic scoring system was developed that allotted 1 point each for these factors.Patients with early HCC could be stratified into 4 distinct prognostic groups (median and 5-year survival,respectively):0 points (97 months,96％),1 point (85 months,76％),2 points (76 months,54％),3 points (56 months,39％) (P ＜0.01).Conclusions The present study emphasized the importance of pathologic staging even in patients with small HCC.Anatomical resection of HCC should be the preferred surgical procedure in cirrhotic patients.

Objective To evalute the clinical application of the new technique of instantaneous wave intensity(WI) through the correlative analysis of instantaneous acceleration wave intensity(W1) and Tei index. Methods The correlation of the intensity of W1 (the apogee of W1) and Tei index of 66 healthy adults were analyzed by Prosound a10 color Doppler ultrasonograph. Results There were positive correlations in pressure and caliber between the common carotid arteries of both sides in 66 cases of healthy adults (P <0.01). There were negative correlations respectively between the pressure of W1 of left and right common carotid arteries and Tei index (P <0.05),and there were the same in caliber (P <0.01), mean pressure (P <0.05) and mean caliber (P <0.01). Conclusions W1 is negatively correlated with Tei index and can be an index in judging the systolic function.

OBJECTIVETo investigate the killing effect of herpes simplex virus thymidine kinase gene expression actuated by the promoter of the vascular endothelial growth factor gene on human hepatocellular tumor cells under hypoxic condition.

METHODSRecombinant adenoviral vectors, AdVEGF-tk and AdVEGF-GFP, were constructed with HSV-tk or GFP under the control of VEGF promoter through AdEasy system. Then GFP expression in hepatoma cell line HepG2 and normal liver cell line L02 transfected with AdVEGF-GFP were observed under fluorescence microscope, and the sensitivity to GCV of the AdVEGF-tk-transfected cells under normoxia or hypoxia condition were monitored by MTT method.

RESULTSGFP expression actuated by VEGF promoter was detected in sporadic L02 cells, but in almost all HepG2 cells after transfected with AdVEGF-GFP. With GCV at 10 micro g/ml and MOI at 100, L02 cells were insensitive to GCV under oxic condition, but more than 70% L02 cells were killed under hypoxic condition. Moreover, HepG2 cells infected with AdVEGF-tk showed the increased GCV sensitivity under hypoxia (over 80% killed) as compared with normoxia (over 60% killed) conditions.

CONCLUSIONHypoxia enhances the GCV sensitivity of human hepatocellular tumor cells infected with recombinant AdVEGF-tk under the control of VEGF promoter.

Adenoviridae ; genetics ; Carcinoma, Hepatocellular ; pathology ; Endothelial Growth Factors ; genetics ; Gene Expression ; drug effects ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Humans ; Hypoxia ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver Neoplasms ; pathology ; Lymphokines ; genetics ; Oxygen ; pharmacology ; Promoter Regions, Genetic ; physiology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo investigate the correlation between midkine (MK) protein expression with local infiltration and metastasis in human hepatocellular carcinoma (HCC).

METHODSImmunohistochemical and Western Blot analysis for MK were performed on samples of tumor tissue and the paratumor tissue from HCC and benign liver tumors.

RESULTSThe overexpression of MK protein determined by immunohistochemical analysis was similar to that by Western Blot analysis. No specific positivity was detected in either benign liver tumor tissue or normal liver tissue, but most of HCC tissue showed a positive reaction to MK immunostain. No correlation between MK expression and other clinicopathological features in MK negative or positive HCC cases was found. Yet, the overexpression rate of MK protein in HCC with intra-hepatic metastasis was significantly higher than that in HCC without intra-hepatic metastasis.

CONCLUSIONIn human hepatocellular carcinoma, MK overexpressed at protein level may very well be closely related to local infiltration and metastasis.

Blotting, Western ; Carcinoma, Hepatocellular ; metabolism ; secondary ; Carrier Proteins ; biosynthesis ; metabolism ; Cytokines ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Metastasis

OBJECTIVETo develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies.

METHODSUsing recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively.

RESULTSA new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy.

CONCLUSIONSGene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Adenoviridae ; genetics ; Adenovirus E1A Proteins ; genetics ; Adenovirus E1B Proteins ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin-12 ; genetics ; Neoplasms ; therapy ; Recombination, Genetic ; Tumor Cells, Cultured ; Virus Replication

OBJECTIVETo study the injection of NKG5SV gene to inhibit growth and metastasis of hepatocellular carcinoma (HCC).

METHODSNKG5SV gene was inserted into retroviral vector pLXSN by normal methods. LacZ gene was used as control. LCI-D20 tumor together with saline, pLXSN-LacZ DNA or pLXSN-NKG5SV was subcutaneously inoculated to the nude mice. Tumor formation rate and tumor size were noted 35 days after inoculation. LCI-D20 tumor was inoculated subcutaneously. Saline, pLXSN-LacZ DNA or pLXSN-NKG5SV was intratumorally injected respectively 10 days after inoculation. Tumor growth was observed 35 days after inoculation. Liver cancer was resected 22 days after intrahepatic inoculation. Saline, pLXSN-LacZ DNA or pLXSN-NKG5SV was respectively injected at incisal margin or intraspleen. Mice were killed 35 days after inoculation to observe tumor recurrence at incisal margin, intrahepatic metastasis and extrahepatic metastasis.

RESULTSTumor formation rate and tumor diameter(cm) were 1.76 +/- 0.11, 1.51 +/- 0.34, 0.33 +/- 0.04 in the control group, LacZ group, NKG5SV group respectively when tumor and different cDNA were inoculated together. Tumor diameter(cm) and weight(g) were 0.87 +/- 0.08, 0.83 +/- 0.05, 0.26 +/- 0.04; 0.43 +/- 0.06, 0.38 +/- 0.04, 0.08 +/- 0.06 in the control group, LacZ group, NKG5SV group respectively when different cDNA were injected into the LCI-D20 tumor. Sites with extrahepatic metastasis nidi, incisal margin recurrence tumor size(cm), intrahepatic metastasis nidi, metastasis involved hepatic lobes in the control group, LacZ group, NKG5SV group were 4.25 +/- 1.48, 4.25 +/- 1.04, 0.63 +/- 0.51; 1.51 +/- 0.27, 1.35 +/- 0.17, 0.81 +/- 0.17; 2.50 +/- 1.41, 2.38 +/- 1.06, 1.25 +/- 0.71; 2.13 +/- 0.99, 2.00 +/- 0.75, 1.38 +/- 0.74 respectively when NK cells were injected at incise margin. They were 4.38 +/- 1.85, 4.25 +/- 1.48, 1.00 +/- 0.75; 1.13 +/- 0.23, 0.97 +/- 0.29, 0.76 +/- 0.16; 2.50 +/- 1.41, 2.05 +/- 1.12, 0; 2.13 +/- 0.83, 1.75 +/- 0.88, 0 respectively when NK cell were injected intrasplenicly.

CONCLUSIONSNKG5SV gene can inhibit HCC growth and postoperative metastasis and recurrence.

Animals ; Antigens, Differentiation, T-Lymphocyte ; Cell Division ; drug effects ; Genetic Therapy ; methods ; Genetic Vectors ; administration & dosage ; genetics ; Humans ; Injections ; Liver Neoplasms, Experimental ; genetics ; pathology ; therapy ; Male ; Mice ; Mice, Nude ; Neoplasm Metastasis ; prevention & control ; Receptors, Immunologic ; genetics ; physiology ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays

Objective To observe changes of the ultrastructure and the cell cycle of hepatoma cells exposed to ultrasound-mediated disruption. Methods Suspensions of Walker-256 hepatoma in vitro exposed to ultrasound disruption with the acoustic power output 0 22 W and the ultrasound frequency 42 kHz were observed by the scanning electron microscopy, the transmission electron microscopy and the flow cytometric analysis of DNA content. Results Cells after ultrasound disruption revealed by scanning electron microscopy exhibited ruptured membrance, disordered organellae and disrupted nucleus, showing typical severe destruction of the cells. Flow cytometric analysis of DNA content demonstrated an increased in the population of cells in the G 2 and M phases of the cell cycle but a decrease in number of cells in the S phase, suggesting an inhibition of the cell synthese and an increased sensitivity to radiation. Conclusions Ultrasound disruption can destroy tumor cells effectively at the cellular level.

Objective To study the mechanism of cytotoxic T lympocytes in the acute rejection of xenogenic liver transplantation(XLT). Methods Hamster to rat orthotopic liver transplantation model was performed with three cuff method. In XLT,lymphocytes including CD8 and CD4 subsets were observed by histology and immunohistochemistry; the expression of perforin and Fas- L was observed by immunohistochemistry; and apoptotic cells of XLT were observed in situ end-labelling of fragmented DNA. Results In the XLT, T cell infiltration firstly ocurred on the 2nd day posttansplantation and located at the portal triads; the infiltrating lymphocytes proliferated increased with time and reached the peak on the 5th to 6th days.Perforin and Fas-ligand were expressed on the 4th day after XLT, and peaked on the 5th to 6th day,and the expression of perforing was still higher than the expression of Fas-ligand. Conclusions T lymphocyte participates in the acute rejection of XLT through the expression of perforin and Fas-ligand, which leads to apoptosis or necrosis of hepatocytes.