Using human epithelial amnion cells in human de-epidermized dermis for skin regeneration.

BACKGROUND: Human amniotic epithelial cells (hAECs) is a desirable reserve of stem cells. Human de-epidermized dermis (DED) retains basic tissue structure and parts of the basement membrane (BM) components at the acelluIar dermal surface, and provides a potential tool for skin regeneration. OBJECTIVE: To evaluate the potential role of hAECs in skin regeneration, we used DED to perform organotypic culture of hAECs to develop organotypic skin. METHODS: HAECs were isolated and cultured. Biological characteristics of hAECs were determined by immunocytochemistry and flow cytometry. To prepare DED, the epidermis was removed and then repeated freeze-thaw cycles. HAECs and fibroblast were seeded onto DED to perform the submerged culture for 3 days and then to be maintained at the air-liquid interface for 14 days to form organotypic culture. To identify whether the obtained DED retain the BM structure and components, the histological characteristics of DED and the BM were detected by immunohistochemistry. To evaluate whether the organotypic skin has similar histological characteristics with normal human skin, the marks of epidermal proliferation and differentiation and basement membrane component were detected by immunohistochemistry. Moreover, cell ultrastructure, cell-cell contact and ultrastructure of BM were examined under the transmission electron microscopy. RESULTS: HAECs has stem-cell characteristics with strong pluripotent Oct-4 and embryonic marker SSEA-4 expression. DED has effectively cleansed the cell components and continuous distributions of laminin and collagen IV. The histological appearance of tissue-engineered skin in vitro has 4 to 9 continuous layers of stratified epithelium and is similar to normal human skin in morphology. Immunohistochemical studies revealed that proliferation and differentiation markers such as Ki67, CK19, CK14, CK10, filaggrin but not CK18 expressed similar pattern characteristics to normal human epidermis. In addition, Periodic acid-Schiff stain showed that a uniform red staining strip located at the epidermal-dermal junction. BM component proteins (type IV collagen and laminin) and cell adhesion protein (desmoglein) were detected by immunohistochemistry in organotypic skin. Ultrastructurally, desmosomes, hemidesmosomes and BM zone (BMZ) were observed in organotypic skin. CONCLUSIONS: Our studies indicate that the hAECs is a promising stem cell source for tissue-engineered skin, and DED with hAECs is a potential application prospects in regenerative medicine.

Effects of heavenly stem-prescription of point selection of needling methods of Ziwu Liuzhu on ischemic cerebrovascular diseases / 中国针灸

<p><b>OBJECTIVE</b>To observe effects of heavenly stem-prescription of point selection of the needling methods of midnight-noon ebb-flow on motorial and neurological functional deficit of ischemic cerebrovascular diseases.</p><p><b>METHODS</b>The patients were randomly divided into a Ziwu Liuzhu group and a channel acupoint selection group. In the channel acupoint selection group, Jianyu (LI 15), Quchi (LI 11), Waiguan (TE 5), Zusanli (ST 36) were selected, combined with selected acupoints by syndrome differentiation. In the Ziwu Liuzhu group, on the basis of the channel acupoint selection group, according to the principle "the acupoints of the yang-channel were opened at yang-day-yang-hour and the acupoints of the yin-channel were opened at yin-day-yin-hour", and acupoints were selected by the day-hour acupoint-opening method of the Ziwu Liuzhu Najia method in Zhenjiu Daquan. The patients in the both groups began to be treated at the Chen hour (7:00-9:00) or Si hour (9:00-11:00) in the morning, 10 times constituting one course. They were treated for 3 courses. Improvement of the motor function symptom cumulative score, Fugl-Meyer (FMA) and improvement of neurological functional deficit were investigated.</p><p><b>RESULTS</b>The FMA score (36.13 +/- 21.80) after treatment was significantly lower than (73.50 +/- 21.53) before treatment (P<0.01) in the Ziwu Liuzhu group, and (54.43 +/- 20.89) after treatment was significantly lower than (62.27 +/- 22.91) before treatment (P<0.05) in the channel acupoint selection group; the neurological functional deficit score (15.40 +/- 9.34) after treatment was significantly lower than (27.17 +/- 10.81) before treatment in the Ziwu Liuzhu group, and (23.97 +/- 1.30) was lower than (27.97 +/- 7.72) before treatment in the channel acupoint selection group. After treatment, FMA and the neurological functional scores significantly improved in the Ziwu Liuzhu group as compared with those in the channel acupoint selection group (P<0.05).</p><p><b>CONCLUSION</b>Ziwu Liuzhu Najia point-selection method can effectively improve both nervous function and motor function in the patient of ischemic cerebrovascular disease.</p>