Effects of Gui Zhi Ma Huang Ge Ban Tang on the TLR7 Pathway in Influenza Virus Infected Mouse Lungs in a Cold Environment.

OBJECTIVE: We wished to investigate the effects of the traditional Chinese medicine Gui Zhi Ma Huang Ge Ban Tang on controlling influenza A virus (IAV) infection and improving inflammation in mouse lungs. METHOD: Mice were maintained in normal and cold environments and infected with IAV by intranasal application, respectively. Real-time quantitative polymerase chain reaction was used to measure mRNA expression of TLR7, myeloid differentiation primary response 88 (MyD88), and nuclear factor-kappa B (NF-κB)p65 in the TLR7 signaling pathway and virus replication in lungs. Western blotting was used to measure expression levels of TLR7, MyD88, and NF-κB p65 proteins. Flow cytometry was used to detect the proportion of T-helper (Th)1/Th2 and Th17/T-regulatory (Treg) cells. RESULTS: Application of Gui Zhi Ma Huang Ge Ban Tang in influenza-infected mice in a cold environment showed (i) downregulation of TLR7, MyD88, and NF-κBp65; (ii) inhibition of transcriptional activities of promoters coding for TLR7, MyD88, and NF-κBp65; (iii) reduction in the proportion of Th1/Th2 and Th17/Treg cells. CONCLUSIONS: Gui Zhi Ma Huang Ge Ban Tang had a good therapeutic effect on mice infected with IAV, especially in the cold environment. It could reduce lung inflammation in mice significantly and elicit an anti-influenza effect by downregulating expression of the key factors in TLR7 signaling pathway.

Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells.

AIM: To compare conventional slow equilibrium cooling and directional freezing (DF) by gauze package for cryopreservation of human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were randomly assigned to conventional freezing (CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide (DMSO), 60% fetal bovine serum (FBS) and 30% Dulbecco's modified Eagle's medium (DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program: precool in 4°C for 30min, -20°C for 1h, and then immersion in -80°C refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in -80°C refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups. RESULTS: There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CF group in adherent rates, morphological changes and proliferative ability. CONCLUSION: In the conventional cryopreserved method, cells are slow equilibrium cooling by steps (4°C, -20°C and finally -80°C), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.

Implantation of Iakymenko keratoprosthesis in patients with severe ocular injury.

AIM: To present the results of implantation of Iakymenko keratoprosthesis in five patients with vascularized corneal leukoma caused by severe ocular injury. METHODS: Iakymenko keratoprosthesis was implanted into 5 eyes of 5 patients: 4 patients were suffered from chemical burns and 1 patient from explosive injury. The preoperative visual acuity ranged from light perception to hand motion. The implantation surgery was composed of two-stage procedures. The follow-up period was from 9 months to 11 years. The outcome measures were visual acuity, retention, and complications of the keratoprosthesis. RESULTS: Vision improvements were achieved in most patients. All keratoprosthesis were retained within the follow-up period. Corneal melting occurred in one patient and fibrous closure in another patient, both of which were successfully treated. Retinal detachment occurred in one patient after surgery. CONCLUSION: Iakymenko keratoprosthesis seems to be a promising alternative for the patients with severe corneal injury, but further investigation is needed to evaluate the role of Iakymenko keratoprosthesis.

[Preliminary study on human bone marrow mesenchymal stem cells differentiation into epithelial-like cells].

OBJECTIVE: To investigate the differentiation potential of human bone marrow mesenchymal stem cells (hMSC) inducing into epithelial-like cells, even corneal epithelial-like cells, and to discuss the plasticity that make hMSC the seed cells used in corneal tissue engineering. METHODS: hMSC were isolated and purified by density gradient centrifugation combined with an attachment culture method and passaged in vitro. hMSC were identified by flow cytometry. The passaged hMSC were planted on fresh pig corneal Bowman's membrane. The expression of CK12, ABCG2 and CK19 in hMSC was identified by immunofluorescence staining. We used in vitro method to obtain a multilayer culture of hMSC. When hMSC formed a monolayer, the cells were inserted to Millicell culture and grew into multilayers by using the air-lifting cultivation methodology. Four weeks later, after fixed and dehydrated, the hMSC were observed under the light microscope after hemotoxylin and eosin (HE) and immunohistochemistry staining. RESULTS: hMSC could be cultured, expanded in vitro, and showed great potential of proliferation. The result of flow cytometry showed that the positive staining percentage was 0.06% for CD45, 0.41% for CD34, 86.43% for CD44, 85.72% for CD29 and 90.72% for CD105. This indicated that hMSC expressed CD44, CD29, CD105 but not CD45 and CD34. After four weeks induction, part of hMSC expressed CK12 and CK19 but not ABCG2. In the in vitro stratification, HE and immunohistochemical staining showed that there were one or two layers epithelial-like cells, even corneal epithelial-like cells after using the air-lifting cultivation. CONCLUSIONS: This study suggests that hMSC have the potential to differentiate into epithelial cells, even corneal epithelial cells. hMSC could be the option of cells used to reconstruct the corneal epithelium by tissue engineering technology.