Cell-penetrating peptide (CPP) can be used in pharmaceutics as a highly efficient drug delivery transporter. In this study, four tumor cell lines (MCF-7, MDA-MB-231, C6, and B16F10) were used to observe the uptake of fluorescein isothiocyanate (FITC) labeled CPP and the effects of time and concentration of CPP on cell penetration was studied. The CPP exocytosis on C6 cell line was observed, and its exocytosis kinetics was described by zero order equation. In addition, low-temperature condition (4 degrees C) and endocytosis inhibitors were utilized to investigate the mechanism of CPP uptake by cells. Low-temperature condition did not show significantly inhibition on CPP uptake. Heparin, a membrane glycoprotein receptor inhibitor, showed strong inhibition effect (only 3%-10% of the control) on CPP uptake. Chlorpromazine, chloroquine and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) showed little effect on CPP uptake. This study indicated that CPP penetration had little selectivity on cell type, but the amount and rate of CPP penetration into cells were related to the type of cell lines. The adsorption of CPP on cell membrane induced by sulfate proteoglycan plays an important role on CPP penetration.

PurposeTo determine the feasibility and accuracy of dual source CT (DSCT) in assessing myocardial delayed-enhancement and left ventricular wall thickness of hypertrophic cardiomyopathy (HCM) in comparison with cardiac magnetic resonance (CMR).Materials and MethodsEighty patients with HCM confirmed by clinical diagnosis were enrolled in the study. DSCT images and CMR images were acquired at the arterial and lag phases. According to 17-segment model provided by American Heart Association, the left ventricular wall thickness and location of delayed-enhancement were verified, and the correlation of these two methods were analyzed in terms of the diagnosis of myocardial delayed enhancement (MDE).Results1360 myocardial segments for 80 patients were assessed. The left ventricular wall thickness determined by DSCT was significantly correlated with MR results (r=0.88,P<0.01). DSCT and MDE showed substantial agreement on per-patient (n=74,Kappa=0.751,P<0.05) and per-segment (n=1238, Kappa=0.746,P<0.01) levels. For dense myocardial delayed enhancement, CT findings were significantly correlated with those of CMR (r=0.89, P<0.01), but CT scan slightly underestimated the lesion scope of fibrosis. Bland-Altman analysis showed that CT and MRI were different in measuring the lesion volume of myocardial delayed enhancement (mean standard deviation was 2.71%).ConclusionThe cardiac CT examination provides comprehensive information in coronary artery and myocardial assessment, and MDE-DSCT is also effective in the diagnosis of myocardial fibrosis in HCM since it can be used in assessing myocardial fibrosis.

Objective To investigate the role of ?-catenin gene in breast tumorigenesis.Methods Mutation and expression of ?-catenin gene in 42 breast cancer tissues were detected by polymerase chain reaction-single strand conformation polymorphism and immunohistochemistry.Results The rate of abnormal expression of ?-catenin in breast cancer tissuse was 59.5%(25/42).Mutation of ?-catenin gene was not detected in breast cancer tissue.Conclusions Abnormal expression of ?-catenin plays an important role in human breast tumorigenesis,but the cause of abnormal expression of ?-catenin is not mutation of ?-catenin gene.

This study was aimed to observe influence on clinical symptoms of Qing-Fei Pei-Yuan Wei-Wan (QFPY-WW) for pulmonary infection HIV/AIDS cases with the syndrome of phlegm-heat obstructing the lung. A total of 141 pulmonary infection HIV/AIDS cases with the syndrome of phlegm-heat obstructing the lung were randomly divided into the treatment group (94 cases) and the control group (47 cases). The treatment group was given basic western medication combined with QFPYWW. The control group was given basic western medication. After 4 weeks treat-ment, observation was made on changes of main symptoms. The results showed that on the 28th day, compared with the control group, there was improvement on symptoms such as cough, breathing and chest tightness (P< 0.05). Com-parison on coughing up phlegm degree showed that the treatment group was significantly better than the control group (P< 0.05). On the 7th day treatment, the treatment group had better effect on lowering body temperature than the control group (P< 0.05). It was concluded that QFPYWW can improve symptoms such as cough, breathing, chest tightness and cough up phlegm among pulmonary infection HIV/AIDS patients.

Antithrombin III (AT III) is the most important anti-clotting substance. Recombinant human antithrombin III (rhAT III) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT III is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT III by isoelectric precipitation and heparin affinity chromatography. First, casein was effectively removed by isoelectric precipitation. rhAT III was further purified by heparin affinity chromatography. In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT III were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied. Under the optimized conditions, the protein recovery of rhAT III was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process is very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.
Animals ; Animals, Genetically Modified ; Antithrombin III ; biosynthesis ; Chromatography, Affinity ; Female ; Goats ; Heparin ; Humans ; Mammary Glands, Animal ; metabolism ; Milk ; chemistry ; Recombinant Proteins ; biosynthesis

OBJECTIVETo investigate the suitable transfection condition of human epidermal cells (hECs) with human epidermal growth factor (EGF) gene by adenovirus vector (Ad-hEGF) and its effects on the biological characteristics of hECs.

METHODShECs were isolated from deprecated human fresh prepuce tissue of circumcision by enzyme digestion method and then sub-cultured. hECs of the third passage were used in the following experiments. (1) Cells were divided into non-transfection group and 5, 20, 50, 100, 150, and 200 fold transfection groups according to the random number table (the same grouping method below), with 3 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in the latter six groups were transfected with Ad-hEGF gene in multiplicities of infection (MOI) of 5, 20, 50, 100, 150, and 200 respectively. The morphology of the cells was observed with inverted phase contrast microscope, and expression of green fluorescent protein of the cells was observed with inverted fluorescence microscope at transfection hour (TH) 24, 48, and 72. (2) Another three batches of cells were collected, grouped, and treated as above, respectively. Then the transfection rate of Ad-hEGF gene was detected by flow cytometer (n=3), the mass concentration of EGF in culture supernatant of cells was detected by enzyme-linked immunosorbent assay (n=6), and the proliferation activity of cells was detected by cell counting kit 8 (CCK8) and microplate reader (n=6) at TH 24, 48, and 72, respectively. (3) Cells were collected and divided into non-transfection group and transfection group, with 6 wells in each group. Cells in non-transfection group were cultured with culture supernatant of cells without transfection, while cells in transfection group were cultured with culture supernatant of cells which were transfected with Ad-hEGF gene in the optimum MOI (50). CCK8 and microplate reader were used to measure the biological activity of EGF secreted by cells on culture day 1, 3, and 5. (4) Cells were collected and divided into non-transfection group and transfection group, with 12 wells in each group. Cells in non-transfection group were not transfected with Ad-hEGF gene, while cells in transfection group were transfected with Ad-hEGF gene in the optimum MOI (50). The expression levels of cytokeratin 14 (CK14) and CK19 of cells were measured by immunofluorescence staining at TH 24. (5) Cells were collected, grouped, and treated as in (4), with 6 wells in each group. At post scratch hour (PSH) 0 (immediately after scratch), 12, 24, and 48, the migration distance of cells was observed and measured with inverted phase contrast microscope. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, and LSD test.

RESULTS(1) At TH 24 and 48, morphology of cells in each transfection group and non-transfection group were similar. Compared with that in non-transfection group, the cell debris increased significantly in 200 fold transfection group at TH 72. At TH 24, 48, and 72, the expression of green fluorescent protein was not seen in cells of non-transfection group, whereas it increased in cells of transfection group over transfection time. (2) The transfection rate of Ad-hEGF gene of cells in each transfection group increased gradually over transfection time. At TH 72, the transfection rates of Ad-hEGF gene of cells in 50-200 fold transfection groups were all above 90%, while the transfection rates of Ad-hEGF gene of cells in non-transfection group, 5, and 20 fold transfection groups were (0.51±0.20)%, (62.44±6.23)%, and (75.00±5.43)% respectively, which were obviously lower than the rate in 50 fold transfection group [(93.12±2.55)%, with P values below 0.01]. The mass concentration of EGF in culture supernatant of cells in each transfection group increased gradually over transfection time. At TH 72, the mass concentration of EGF in culture supernatant of cells in 50 fold transfection group was obviously higher than that in each of the other groups (with P values below 0.01). The proliferation activity of cells in each group at TH 24 and 48 was similar (with P values above 0.05). At TH 72, the proliferation activity of cells in 200 fold transfection group was obviously lower than that in other groups (with P values below 0.05). (3) On culture day 1, the biological activity of EGF secreted by cells in two groups was similar (P>0.05). On culture day 3 and 5, the biological activity of EGF secreted by cells in transfection group were obviously higher than that in non-transfection group (with P values below 0.01). (4) At TH 24, the expression levels of CK14 and CK19 of cells in transfection group were higher than those in non-transfection group. (5) The width of scratch in two groups was nearly the same at PSH 0. At PSH 12-48, the migration distance of cells in transfection group was obviously longer than that in non-transfection group (with P values below 0.01).

CONCLUSIONSThe suitable range of MOI of hECs transfected with Ad-hEGF gene is 50-150, and 50 is the optimum. hECs transfected with Ad-hEGF gene with MOI 50 can effectively express the EGF gene and keep its good abilities of proliferation, differentiation, and migration, as well.

Adenoviridae ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; EGF Family of Proteins ; genetics ; metabolism ; Epidermis ; cytology ; Genetic Vectors ; Humans ; Keratins ; metabolism ; Male ; Transfection

A major challenge facing photodynamic therapy (PDT) is that the activity of the immune-induced infiltrating CD8⁺ T cells is subject to the regulatory T lymphocytes (Tregs), leaving the tumor at risk of recurrence and metastasis after the initial ablation. To augment the antitumor response and reprogram the immunosuppressive tumor microenvironment (TME), a supramolecular photodynamic nanoparticle (DACss) is constructed by the host-guest interaction between demethylcantharidin-conjugated β-cyclodextrin (DMC-CD) and amantadine-terminated disulfide-conjugated FFVLGGGC peptide with chlorin e6 decoration (Ad-ss-pep-Ce6) to achieve intelligent delivery of photosensitizer and immunomodulator for breast cancer treatment. The acid-labile β-carboxamide bond of DMC-CD is hydrolyzed in response to the acidic TME, resulting in the localized release of DMC and subsequent inhibition of Tregs. The guest molecule Ad-ss-pep-Ce6 can be cleaved by a high level of intracellular GSH, reducing photosensitizer toxicity and increasing photosensitizer retention in the tumor. With a significant increase in the CTL/Treg ratio, the combination of Ce6-based PDT and DMC-mediated immunomodulation adequately achieved spatiotemporal regulation and remodeling of the TME, as well as improved primary tumor and in situ lung metastasis suppression with the aid of PD-1 antibody.