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题名:Engineered Insulin Secretion from Neuroendocrine Cells Isolated From Human Thyroid.
作者:THULE PM; JIA D; SAFLEY S; GORDON K; BARBER G; YI H; NALLI S; ONDERCI M; SHARMA J; SHIRES J; WEBER CJ;
来源:World J Surg. 2014 Feb 19. [ IF= 0.00 ] ]
URL :10.1007/s00268-014-2457-7
日期:20140317
摘要:BACKGROUND: Insulin-secreting beta-like cells are vulnerable to diabetic
autoimmunity. We hypothesized that human thyroid neuroendocrine (NE) cells could
be engineered to secrete human insulin, be glucose-responsive, and avoid
autoimmunity. METHODS: Collagenase-digested thyroid tissue was cultured and
subjected to size-based fluorescence-activated cell sorting. Insulin secretion
and storage in NE cells transduced with viral vectors carrying an insulin
sequence was assessed by enzyme-linked immunosorbent assay (ELISA) and immunogold
transmission electron microscopy (TEM). Baseline mRNA expression was assessed by
Illumina expression array analysis. Transduction with retrovirus expressing
transcription factors PDX1, NGN3, MAFA, or HNF6 altered mRNA expression in a
custom polymerase chain reaction (PCR) array. Gastrin-releasing peptide (GRP) in
conditioned medium and cell lysates was determined by reverse transcription
(RT)-PCR, ELISA, and immunohistochemistry. RESULTS: Isolation yielded an average
of 2.2 x 106 cells/g thyroid tissue, which stained for calcitonin/calcitonin
gene-related protein, expressed genes consistent with NE origins, and secreted
GRP. Transduced cells secreted 56 % and retained 48 % of total insulin produced.
Immunogold TEM revealed insulin in secretory vesicles. PDX1, NGN3, and MAFA
overexpression increased expression of genes typical for hepatocytes and beta
cells. Overexpression of HNF6 also increased the message of genes critical for
glucose sensing. CONCLUSIONS: Human thyroid NE cells can produce human insulin,
fractions of which are both secreted and retained in secretory granules.
Overexpression of HNF6, PDX1, or NGN3 enhances expression of both hepatocyte and
beta cell typical mRNAs, including the message of proteins critical for glucose
sensing. These data suggest that reimplantation of engineered autologous NE cells
may develop as a viable treatment for diabetes mellitus type 1.

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