The aim of the study is to establish a platform to deliver therapeutic proteins into target cells through a polyarginine-based cell penetrating peptide. To facilitate the expression of therapeutic proteins, a pSUMO (Small Ubiquitin-like Modifier)-R9-EGFP (enhanced green fluorescence protein) prokaryotic expression vector was constructed. After induction, the fusion protein SUMO-R9-EGFP was efficiently expressed. To validate the cell penetrating ability of the fusion protein, HepG2 cells were incubated with the purified R9-EGFP or EGFP protein as control, internalization of the fluorescent proteins was examined by either flow cytometry or confocal microscopy. The result obtained by flow cytometry showed that the R9-EGFP fusion protein could efficiently penetrate into the HepG2 cells in a dose and time-dependent manner. In contrast, the fluorescence was barely detected in the HepG2 cells incubated with EGFP control. The fluorescence intensity of the R9-EGFP treated cells reached plateau phase after 1.5 h. The result obtained by confocal microscopy shows that R9-EGFP efficiently entered into the HepG2 cells and was exclusively located in the cytoplasm, whereas, no fluorescence was detected in the cells incubated with the EGFP control. The heparin inhibition experiment showed that heparin could inhibit penetrating effect of the R9-EGFP protein by about 50%, suggesting that the penetrating ability of the fusion protein is heparin-dependent. In summary, the study has established a platform to deliver therapeutic proteins into target cells through a polyarginine-based penetrating peptide.
Cell-Penetrating Peptides ; biosynthesis ; genetics ; pharmacology ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Peptides ; genetics ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

AIM: To observe the expressions of aquaporin 2 (AQP2) in kidney tissues and the contents of endotoxin (ET), interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α) in serum in emphysema model rats, and to investigate the relationship between lungs and kidney in humoral metabolism. METHODS: The rats of emphysema were treated by injecting lipopolysaccharide into the trachea with cigarette smoking. Immunohistochemistry and Western blotting analysis were used to observe the expression of AQP2 in kidney tissues. RT-PCR was applied to detect the expression of AQP2 mRNA in kidney tissues. Blood sample and lung tissue were taken and the levels of ET, IL-1β and TNF-α were measured by radioimmunoassay. RESULTS: AQP2 expression in the kidney tissue in model group was greater than that in control group, and the expression of AQP2 mRNA showed the same results (P<0.01). ET, IL-1β and TNF-α levels in serum and lung tissue in model group were markedly higher than those in control group (P<0.01). CONCLUSION: In the emphysema model rats, AQP2 expression is up-regulated in the kidney tissue. The mechanism of emphysema may be related to increasing the levels of ET, IL-1β and TNF-α in the serum and lung tissue obviously.

OBJECTIVE: Anatomic variation of renal artery existed in donor kidney, and whether the variation would affect clinical effects is unconfirmed. This paper is aimed to study the outcomes of in vitro reconstruction of malformed renal blood vessels on outcome of kidney transplantation. METHODS: Patients treated with kidney transplantation at Department of Urology, Lanzhou General Hospital of Chinese PLA were selected; including 27 cases underwent kidney transplantation with malformed blood vessels. Seventeen of them had accessory renal artery, 10 of them with two or three renal arteries. Additional 22 patients transplanted with normal kidney during the same period were selected as the control group. Bench surgery with microsurgical techniques was employed for the repair of the vessels, which included 11 cases of end to end anastomosis of arteries, 6 cases of end to side anastomosis of arteries, 6 cases of side to side conjoined anastomosis of arteries, and 4 cases of renal artery lengthening with an interposition of donor or recipient iliac artery. The hemorrhage, hypertension, renal arterial stenosis, delayed graft function, incidence rate of acute rejection, and the serum creatinine level were followed up.RESULTS: Totally 49 cases were received a mean 2-year follow-up, and no death occurred in both groups. Two cases in the experimental group, and 3 cases in the control group were suffered hypertension or hypertension aggravated, the difference had no significant (P =0.673). No arterial stenosis occurred during the follow up. In the experimental and control groups, the incidence of delayed graft function was 20% and 14%, the incidence of acute rejection was 13% and 5%, and mean serum creatinine at 2 year was (119±11) and (127±8) μmol/L, respectively, the difference was not significant between two groups (P=0.179, 0.385, 0.658).CONCLUSION: Donor kidneys with malformed vascular can be used for transplantation after bench reconstruction, which do not influence the outcome of kidney transplantation.

OBJECTIVE: Cisplatin is a widely used chemotherapeutic agent in the treatment of cancers in clinic; but it often induces adverse effects on ovarian functions such as reduced fertility and premature menopause. Mesna could attenuate the cisplatin-induced ovarian damages; however, the underlying mechanism is still unknown. This study aimed to figure out the underlying mechanism of the protection of mesna for ovaries against cisplatin therapy in cancers. METHODS: We performed female adult Sprague-Dawley rats into normal saline control (NS), low-dose cisplatin (CL), high-dose cisplatin (CH), CL plus mesna (CL+M), and CH plus mesna (CH+M) groups and detected anti-Mullerian hormone (AMH)-positive follicle, oxidative stress status and anti-oxidative capability in ovaries. RESULTS: AMH-positive follicles were significantly decreased after cisplatin administration, which was significantly reversed when mesna was co-administered with cisplatin. The end product of lipid peroxidation, malondialdehyde (MDA), was significantly increased, but the anti-oxidative enzymatic activity of superoxide dismutase (SOD) and glutathione (GSH) were significantly decreased in cisplatin groups when compared with NS group. In contrast, after co-administration of cisplatin with mesna, MDA was significantly decreased whereas the activity of SOD and the concentration of GSH were increased. Moreover, mesna did not decrease the anti-tumor property of cisplatin in HePG2 cell lines. CONCLUSION: Cisplatin damages the granulosa cells by oxidative stress to deplete the ovarian reserve and mesna could protect ovarian reserve through anti-oxidation. These results might highlight the mechanism of the protection of mesna for ovarian reserve and open an avenue for the application of mesna as a protective additive in cisplatin chemotherapy in clinical practise.
Adult ; Animals ; Anti-Mullerian Hormone ; Cisplatin ; Female ; Fertility ; Glutathione ; Granulosa Cells ; Hep G2 Cells ; Humans ; Lipid Peroxidation ; Malondialdehyde ; Menopause, Premature ; Mesna ; Ovary ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase