CN100448483C - Treatment for diabetes - Google Patents

Abstract

Proliferating pancreatic islet cells obtained by the method of isolating a population of cells that preferably includes predominantly islet precursor cells that express one or more marker associated with an islet precursor cell and providing the precursor cells with one or more a pancreatic differentiation agent so that a population of cells is obtained that has a high proportion of cells with phenotypic characteristics of functional pancreatic islet beta-cells. Optionally, the precursor cells are pretreated by providing them with one or more cell expansion agent to increase the number of cells in the population prior to differentiation. The pancreatic differentiation agent composition comprises a gastrin/CCK receptor ligand, e.g., a gastrin, in an amount sufficient to effect differentiation of pancreatic islet precursor cells to mature insulin-secreting cells. The cell expansion agent composition comprises one or more epidermal growth factor (EGF) receptor ligand in an amount sufficient to stimulate proliferation of the precursor cells. The methods of treatment include transplanting either undifferentiated precursor cells and providing the pancreatic differentiation agent either alone or in combination with the cell expansion agent in situ, or transplanting the functional pancreatic islet beta-cells into the patient. The pancreatic islet beta-cells can be used for drug screening, and replenishing pancreatic function in the context of clinical treatment.

Description

Treatment of diabetes

Preface

Invention field

Present invention relates in general to the cytobiology field of pancreas precursor, and the method that is used to obtain sophisticated islet cells.More particularly, the present invention relates to express the human stem cell of one or more labellings relevant or the directed differentiation of other islets of langerhans precursors becomes functional pancreas beta cell with the islets of langerhans precursor, comprise in gastrin-receptor part and the EGF receptors ligand one or both are provided, and relate to the method for the pancreatic diseases that comprises diabetes that described cell is used for the treatment of the individuality of this treatment of needs.The example of described method is that (a) provided the gastrin-receptor part external to human pancreatic island cell before transplanted cells, so that stimulate insulin to produce, described cell is optional to provide EGF receptors ligand, the quantity of described cell so that increase, (b) employing is transplanted human pancreatic island cell and with uniting of one or both interior therapeutics in gastrin-receptor part and the EGF receptors ligand mouse model system of diabetes being carried out treating diabetes in the body, is promoted propagation and/or insulin production so that pass through the islet cells and islet cell.

Background

Diabetes are one of modal endocrinopathyes that occur among all age group and the crowd.Except clinical onset rate and fatality rate, the economy consumption of diabetes is huge, and is only annual just above 90,000,000,000 dollars in the U.S., and estimate that the popular of diabetes will increase more than the twice by 2010.

The diabetes that have two kinds of principal modes: insulin-dependent (1 type) diabetes (IDDM), it accounts for the 5-10% of all cases, and non-insulin-depending type (2 type) diabetes (NIDDM), and it roughly accounts for 90% of case.The increase at type 2 diabetes mellitus and age is relevant, but, exists by diagnosis and suffers from the trend that the youthful quantity of NIDDM increases, promptly so-called youthful ripe outbreak diabetes (MODY).In 1 type and 2 type cases, there is the forfeiture of insulin secretion, this phenomenon is that the defective by the destruction of beta cell in the pancreas or insulin secretion or production causes.In NIDDM, the patient is usually according to best diet, and body weight reduces and the scheme begin treatment of motion.When no longer providing suitable metabolism to control, these measures begin Drug therapy.Initial Drug therapy comprises the sulphur urea, and it can stimulate the beta cell insulin secretion, but, can also comprise biguanide, β-Pu Tangganmei inhibitor, thiazolidenediones and therapeutic alliance.But, development character that it should be noted that the described disease mechanisms that plays a role in type 2 diabetes mellitus is unmanageable.The diabetics of all Drug therapys surpassed 50% and showed relatively poor glycemic control in 6 year, and no matter used which type of medicine.The a lot of people of insulinize quilt think to treat the last-resort of type 2 diabetes mellitus, and there is the psychology of resisting the use insulin in the patient.Diabetic complication comprises influences retina, the complication of the little blood vessel in kidney and the nerve (microvascular complication) and the influence to heart, the complication of the trunk of brain and lower limb blood supply (trunk complication).The diabetes microvascular complication is a newly-increased blind main cause among 20-74 year crowd, and accounts for 35% of all new end-stage renal disease cases.Diabetes above 60% are influenced by neuropathy.In the U.S., diabetes account for 50% of all atraumatic amputation, mainly caused by diabetes trunk disease, and the mortality rate of the coronary artery disease of diabetics are higher 2.5 times than non-diabetic.Hyperglycemia is considered to induce and to quicken the progress of diabetes microvascular complication.Adopt various existing therapeutic schemes, all can not suitably control hyperglycemia, therefore, can not prevent or alleviate the progress of diabetic complication.

Islets of langerhans is to be formed by the endothelial stem cell growth that is present in the fetus conduit pancreas endotheliocyte, and it also comprises the pluripotent stem cell that can develop into exocrine pancreas.Teitelman and Lee, Developmental Biology, 121:454-466 (1987); Pictet and Rutter, embryo's endocrine embryo's growth is referring to Endocrinology, Handbook ofPhysiology, ed.R.O.Greep and E.B.Astwood (1972), American Physiological Society:Washington, D.C., p.25-66.The islets of langerhans growth is to be undertaken by the independently stage of development between embryo's stage of incubation, and its can be interrupted by violent variation.The initial stage is original differentiation state, it is characterized in that pluripotent stem cell is shaped into islet cells system, as by original noble cells to insulin and glucagon expression embodied.Described original noble cells comprises the islets of langerhans precursor of a group typing, and these cells can only be expressed low-level islets of langerhans specific gene product, and lack the cell differentiation of sophisticated islet cells.Pictet and Rutter, the same.About 16 days, original differentiation pancreas has begun the stage of quick growth and differentiation at mouse pregnancy, it is characterized in that the increase of the hundred times of the cell differentiation of islet cells and islets of langerhans expression of specific gene.On the histology, islets of langerhans forms (new life) because the islets of langerhans bud of breeding from described ductus pancreaticus becomes obviously (nesidioblast propagation).Before being about to childbirth, the islets of langerhans speed of growth slows down, and islet neogenesis and nesidioblast propagation become more obvious.Meanwhile, islets of langerhans has reached the state of differentiation fully, and the insulin gene with top level is expressed.Therefore, similar with a lot of organs, the end of cell differentiation is relevant with the reproducibility potentiality that weakened; The one-tenth human pancreas of described differentiation does not possess regeneration potential or the multiplication capacity identical with developmental pancreas.

Carry out late period because the differentiation of the precursor of original differentiation is the fetal development at pancreas, regulate the factor of islets of langerhans differentiation and may in pancreas, express in this stage.One of described gene of expressing between the period of development at islets of langerhans coding a kind of gastrointestinal peptide, i.e. gastrin.Although gastrin works with the excretory form of gastric hormone modulability acid in the adult, the main position that gastrin is expressed in the embryo is an islets of langerhans.Brand and Fuller, J.Biol Chem., 263:5341-5347 (1988).The expression of gastrin in islets of langerhans is instantaneous.The islets of langerhans precursor that it is confined to original differentiation forms the stage of the islets of langerhans of differentiation.Although the pancreas gastrin is unknown in the developmental effect of islets of langerhans, some clinical observation shows that gastrin is as follows in the developmental rule of this islets of langerhans.For example, relevant with the nesidioblast hyperproliferative disorder by gastrin expressivity islet cell tumor with the hypergastrinemia that atrophic gastritis is caused.Be similar to situation about occurring in the embryo's islets of langerhans in differentiation.Sacchi etc., Virchows Archiv B, 48:261-276 (1985); With Heitz etc., diabetes, 26:632-642 (1977).In addition, in the case of neonate nesidioblastosis, put down in writing the unusual existence of pancreas gastrin already.Hollande etc., Gastroenterology, 71:251-262 (1976).But, these two kinds of discoveries all do not have to set up cause effect relation between nesidioblastosis and gastrin stimulation.

Therefore, it is valuable that evaluation can stimulate islet cells propagation and/or regenerated preparation, for use in treating early stage IDDM and being used for preventing the beta cell of NIDDM to lack.

Pertinent literature

There have three kinds of somatomedin and fetal pancreas to grow to be relevant, gastrin, transforming growth factor (TGF-α) and epidermal growth factor (EGF) (Brand and Fuller, J.Biol.Chem.263:5341-5347).The TGF-α that overexpression is independent or the transgenic mice of gastrin can not show active islet cells growth, but, can express the remarkable increase (Wang etc., (1993) J Clin Invest 92:1349-1356) that two kinds of genetically modified mices show islet cells group.Bouwens and Pipeleeis (1998) Diabetoligia 41:629-633 has reported and had a high proportion of beta cell that sprouts in the normal adult human pancreas, and finds that 15% of all beta cells are single units.One beta cell kitchen range is uncommon in adult (stimulation) pancreas in rat; Wang etc. ((1995) Diabetologia 38:1405-1411) have reported the frequency that is approximately total β cell quantity 1%.

In following document, disclosed the insulin dependent/non-dependent of type 1 diabetes patient after tunicate islet transplantation: Soon-Shiong etc. (1994) Lancet 343:950-51.Also can be referring to (on June 15th, 1998) Transplantation 65 (11): 1510-1512 such as Sasaki; Zhou etc. (in May, 1998) Am J Physiol 274 (5Pt1): C1356-1362; Soon-Shiong etc. (June nineteen ninety) Postgrad Med 87 (8): 133-134; Kendall etc. (in June, 1996) Diabetes Metab 22 (3): 157-163; Sandler etc. (in June, 1997) Transplantation 63 (12): 1712-1718; Suzuki etc. (in January, 1998) CellTransplant 7 (1): 47-52; Soon-Shiong etc. (in June, 1993) Proc Natl Acad SciUSA 90 (12): 5843-5847; Soon-Shiong etc. (in November, 1992) Transplantation54 (5): 769-774; Soon-Shiong etc. (in October, 1992) ASAIOJ38 (4): 851-854; Benhamou etc. (in June, 1998) Diabetes Metab 24 (3): 215-224; Christiansen etc. (in December, 1994) J Clin Endocrinol Metab 79 (6): 1561-1569; Fraga etc. (in April, 1998) Transplantation 65 (8): 1060-1066; Korsgren etc. (1993) Ups JMed Sci98 (1): 39-52; Newgard etc. (in July, 1997) Diabetologiz 40Suppl 2:S42-S47.

Summary of the invention

The patient's who is used for the treatment of needs treatments needs the diabetes or the method and composition of other pancreatic diseases are provided, wherein, provide in gastrin-receptor part and the EGF receptors ligand one or both, so that stimulation pancreatic cell regeneration and/or new life.Described compositions comprises the islet cells of a group propagation that obtains by the following method: separate a group cell, and one or more pancreas differentiation agents are provided for described precursor, so that obtain a group functional islets cell.One or more cell expansion agent are provided randomly for described precursor,, normally before handling, provide with differentiation agent so that increase the quantity of cell in the described colony.Preferably described cell mass is carried out enrichment, so that comprise the islets of langerhans precursor of one or more labellings relevant of expression of higher percentage ratio with the islets of langerhans precursor, therefore and has an a high proportion of cell with phenotypic characteristic of functional islets beta cell, described phenotypic characteristic comprises the morphological feature of beta cell, express the surface markers feature of beta cell, and have enzymatic important concerning pancreatic function and biosynthesis activity.Described pancreas differentiation agent compositions comprises gastrin/cck receptor part, for example, gastrin, its consumption is enough to realize that the islets of langerhans precursor is divided into sophisticated insulin secretory cell.Described cell expansion agent composition comprises one or more epidermal growth factors (EGF) receptors ligand, and its consumption is enough to stimulate described precursor propagation.Randomly these two kinds of reagent are used for increasing and one or two of differentiation step.Described Therapeutic Method comprises to be transplanted to undifferentiated precursor in the host animal body, and provide the pancreas differentiation agent separately or with the cell expansion agent combination in vivo, or exsomatize one or both receptors ligands are provided, then the functional islets beta cell is transplanted in the host animal body.This system provides the source that is used for multi-purpose functional islets beta cell, as drug screening with in clinical treatment, particularly compensates pancreatic function in the diabetes clinical treatment.

The accompanying drawing summary

Fig. 1 represents the influence to the glucose tolerance of the inductive diabetes Wistar rat of streptozotocin of TGF-α and gastrin, and this rat is handled with the 10 day time of combination peritoneal injection (solid purple rectangle) of PBS (filled black rhombus) or TGF-α and gastrin every day.

Fig. 2 represents that TGF-α and gastrin handle the influence that three groups of Zucker rats of handling and corresponding PBS is contrasted (every group of n=6) middle beta cell new life, as disclosed in the embodiment 4.Light blue post is represented thin TFG+ gastrin, and the reddish violet post is represented fat TGF+ gastrin, and yellow post is represented fat PBS contrast, and the navy blue post is represented pre TFG+ gastrin, and the purple post is represented thin PBS contrast.Compare with the control animal that PBS handles, TGF-α and gastrin can obviously improve the relative scale of the single beta cell kitchen range in all seminar.The 4th group obviously different with first and second groups (p＜0.0041) with the 5th group (p＜0.0015).

Fig. 3 is illustrated in TGF-α and gastrin in the fat Zucker rat of lean and fat and handles influence to beta cell new life.By adding up total beta cell respectively and newborn single beta cell kitchen range carries out quantitatively beta cell new life, and represent with the percentage ratio form of total beta cell of adding up.The percentage ratio of single beta cell kitchen range is 10.5 ± 0.9 in the thin Zucker rat of handling with combinations of. growth factors, and corresponding therewith is to be 3.9 ± 1.1 (p=0.004) (Fig. 3 A and 3B) in corresponding PBS contrast.In fat Zucker rat, the percentage ratio of the single beta cell kitchen range in pretreated group is 8.7 ± 1.3, and the percentage ratio in corresponding matched group is 4.2 ± 1.1 (p=0.0015) (Fig. 3 C and 3D).Fig. 3 E is the image of 400 times of the amplifications of catheter area shown in Fig. 3 C (representing by arrow), and the clear and definite evidence that provides the beta cell that contains insulin from the newborn peculiar ductal epithelial cell of beta cell to sprout.

Fig. 4 represents to have reduced fasting blood glucose level with G1 treatment in chronic diabetes insulin-dependent NOD mice, and suppresses death after stopping insulinize in 14 days.

Fig. 5 represents to have reduced fasting blood glucose level with EGF treatment on chronic diabetes insulin-dependent NOD mice, and suppresses death after stopping insulinize in 14 days.

Fig. 6 represents to have suppressed with E1 or G1 treatment the raising of fasting blood glucose level in the NOD of the diabetes of suffering from nearest outbreak mice.

Fig. 7 represents to have improved with E1 or G1 treatment the intravital pancreas insulin content of NOD mice of the diabetes of suffering from nearest outbreak.

Fig. 8 is illustrated in the result that the EGF/ gastrin is handled in the diabetic mice.Fig. 8 A is the result's of expression glucose tolerance test a line chart, this curve shows blood glucose (left side curve) or blood plasma people C-peptide (right side graph) on vertical coordinate, it is the function of time (reaching 120 minutes most) of illustrating on abscissa, this test is with having transplanted people's islets of langerhans and with gastrin/EGF (EGF, 30 μ g/kg, and gastrin, 1000 μ g/kg, filled symbols) the NOD-Scid mice of Chu Liing, or carry out with the control mice of only accepting excipient (hollow).Curve representation gastrin/the EGF on right side has improved the insulin secretion reaction of human tissue.Fig. 8 B is the bar diagram that the content that is illustrated in the people C-peptide in the mice plasma that the EGF/ gastrin handled is higher than the intravital content of handling at excipient of mice.

Fig. 9 be expression use EGF+ gastrin (light grey post) or excipient (white post) or in advance islet transplantation (Dark grey post) implantation in the NOD-Scid mice body of people's islets of langerhans with the bar diagram of microgram/insulin content that the graft form is represented.Data representation is compared with untreated NOD-Scid mice, and gastrin/EGF has improved the insulin content of implanting the intravital people's islets of langerhans of handling of NOD-Scid mice.

Figure 10 is the beta cell percentage ratio (left side curve) in the intravital people's islets of langerhans of implantation mice identical with Fig. 2 and the bar diagram of beta cell sum (right side graph).The result shows that gastrin/EGF can stimulate the beta cell new life who implants the intravital human islets of langerhans of handling of NOD-SCID mice.

Figure 11 is in the intravital complete islet transplantation thing of NOD-SCID mice or one group of microphotograph of the insulin positive cell (dying dead color) in isolating islet transplantation thing cell.Data show, gastrin/EGF can induce the raising of insulin positive cell content of people's islets of langerhans of transplanting.

Figure 12 will link together in expression of the PDX-1 in the cell of handling and insulin expression.Figure 12 A is the co of expression PDX-1 dyeing human pancreatic island cell and PDX-1 and in one group of microphotograph of gastrin/EGF-and the insulin expression in the cell that excipient was handled.Figure 12 B is illustrated in people's islets of langerhans is implanted the bar diagram that PDX-1 expresses after 8 weeks in the NOD-SCID mice body, during this period, handles described mice with gastrin/EGF or with excipient.

Figure 13 is one group of line chart of the result of expression glucose tolerance test.Blood sugar content (Image to left) or blood plasma people C-peptide (Image to right) are illustrated on the vertical coordinate, function as the time on the abscissa (can reach 120 minutes), described test is with having transplanted people's islets of langerhans and with low dosage gastrin/EGF (EGF, 30 μ g/kg, and gastrin, 30 μ g/kg; The rectangle symbol) or the NOD-SCID mice of handling with excipient (circle symbol) carry out.Data show that even under low dosage, gastrin/EGF also can improve the insulin secretion reaction of people's tissue.

Detailed description of preferred embodiments

The invention provides the patient's who is used for the treatment of the needs treatment the diabetes and the method and composition of other degeneration pancreatic diseases, gastrin/cck receptor the part that provides such as gastrin is provided, and/or such as the EGF receptors ligand of TGF-α or EGF, or the two combination, its consumption is enough to realize that the islets of langerhans precursor is divided into sophisticated insulin secretory cell.When systematicness ground applying said compositions, normally use preferably acceptable carrier medium-sized vein injection on the physiology by injection.When described composition in situ was expressed, the islets of langerhans precursor was stripped or transforms with one or more expression of nucleic acid constructs that are present in the expression vector in vivo that described expression vector can provide the expression of receptors ligand in the islets of langerhans precursor that needs.As a kind of example, described expression construct comprises the coded sequence of cck receptor part, as preprogastrin peptide precursor coded sequence, it is after expressing, be processed to gastrin, or the coded sequence of the EGF receptors ligand of coding such as TGF-α, and transcribe and the translational control district, it provides the expression of islets of langerhans precursor.Described transcription regulatory region can be composing type or induction type, for example, by improving concentration of glucose in the cell, as transcription regulatory region from insulin gene.Conversion is carried out with any suitable expression vector, for example, and adenovirus expression carrier.When described conversion is stripped carrying out, the cell that transformed is implanted in the diabetics body, for example, use the scrotum.

In addition, with the gastrin/cck receptor part and/or the EGF receptors ligand ex vivo treatment islet cells of sufficient amount, so that increased the quantity of precursor pancreas beta cell before in implanting the diabetics body.As required, after the amplification, break up precursor pancreas beta cell group in culture in exsomatizing before transplanting, this realizes by allowing described cell contact with the gastrin-receptor part at least.No matter increasing and/or breaking up is to carry out before or after transplanting, and carries the islets of langerhans precursor in processing, as stem cell or express before the cell of one or more labellings of vessel cell of CK 19 cell as described in the optional enrichment.

The invention provides and be better than the existing advantage that is used for the treatment of the therapeutic scheme of diabetics.Stimulate the adult pancreatic cell so that regenerated mode by providing, it be not only reduce or or even to eliminate conventional medicament treatment (2 type) or insulinize (1 type and 2 types) necessary, and the maintenance of euglycemia level can also alleviate the diabetic complication that some has more disabling property.Amplification by before or after transplanting, using islet tissue or other islets of langerhans precursors and in the differentiation one or both, particularly combine, the means that reduce the islets of langerhans quantity of transplanting needed rareness are provided with the precursor group's of the islets of langerhans precursor that comprises larger proportion enrichment.In addition, be not only the transmutability of the cell mass that is used to transplant that reduces by the inventive method, but also have the increase of reproducibility of the functional characteristic of described transplanted cells.Another advantage of the present invention is, can weaken immunologic rejection, for example, and the xenotransplantation on Pancreas Sus domestica island.

In this article, term " gastrin/cck receptor part " comprises the chemical compound that can stimulate gastrin/cck receptor.The example of described gastrin/cck receptor part comprises various forms of gastrins, as gastrin 34 (big gastrin), and gastrin 17 (little gastrin) and gastrin 8 (miniature gastrin); Various forms of cholecystokinins, as CCK58, CCK 33, and CCK 22, CCK12 and CCK 8; And other gastrins/cck receptor part, they can independently or with the combination of EGF receptors ligand induce the differentiation of cell in the ripe pancreas, so that form the islet cells of excreting insulin.The active analogue thereof that also relates to above-claimed cpd, segment and other modified forms, the peptide and non-peptide agonists or the partial agonist that comprise gastrin/cck receptor, as A71378 (Lin etc., Am.J.Physiol.258 (4Pt1): G648,1990), it self or with the combination of EGF receptors ligand after can induce cell differentiation in the ripe pancreas form can excreting insulin islet cells.Interested especially is the gastrin derivant, and it has the leucine that replaces No. 15 locational methionines.Referring to U.S. Pat PN10/044,048, open day is on July 25th, 2002, the content of this patent is done this paper reference by receipts.Gastrin/cck receptor part also comprises can strengthen the excretory chemical compound of endogenous gastrin, cholecystokinin or come the similar active peptide of having of self-organizing storing position.The example of described peptide is, as EGF and its analog and fragment, and non-peptide micromolecule, as omeprazole, its can gastric acid inhibitory secretion and/or increases the quantity of gastrin/cck receptor, and can strengthen the soybean trypsin inhibitor that CCK stimulates.

In this article, term " EGF receptors ligand " comprises the chemical compound that can stimulate the EGF receptor, when also being upset in identical or adjacent tissue or in individuals with same the time with convenient gastrin/cck receptor, induces the new life of insulin production islet cells.The stimulation of gastrin/cck receptor can directly be carried out by gastrin/cck receptor part is provided, or for example, by endogenous and/or exogenous factor in vivo the gastric acid inhibitory secretion carry out indirectly.The example of EGF receptors ligand comprises EGF1-53 and its fragment and active analogue thereof, comprises EGF1-48, EGF1-52, EGF1-49. for example, referring to U.S. Pat PN 5,434,135.Other interested analog comprise having the EGF that length is the aminoacid sequence of X, X is at least 48 and be no more than 53 integer, described sequence (i) be with from No. 1 position of human EGF to the aminoacid sequence part of the X-1 position of human EGF homology and (ii) have and be different from the amino acid residue that is present on the human EGF substantially in the X position.Interested especially is the analog of human EGF, and wherein, X is 51, and, be aminoacid except glutamic acid wherein at the locational amino acid residue of X, for example, neutral amino acid, hydrophobic amino acid, or charged aminoacid.When X was 51, interested replacement comprised agedoite, glutamine, alanine and serine (referring to PCT/US02/233097, open day is on May 15th, 2003, and its content is done this paper reference by receipts).Other examples of EGF receptors ligand comprise TGF-α receptors ligand (1-50) and its fragment and active analogue thereof, comprise 1-48,1-47 and other EGF receptors ligands, as amphiregulin and poxvirus somatomedin, and other EGF receptors ligands, the synergistic activity that they have with gastrin/the cck receptor part is identical.Comprising the active analogue thereof of above-mentioned substance, fragment and modified forms.Relevant further background can be referring to Carpenter and Wahl, Chapter 4in Peptide Growth Factors (Eds.Spornand Roberts), Springer Verlag, (1990).

Main aspect of the present invention is the method for diabetes that is used for the treatment of the individuality of needs treatments, comprise the compositions that comprises gastrin/cck receptor part and/or EGF receptors ligand to described individuality is provided, its consumption is enough to realize that the islets of langerhans precursor is divided into sophisticated insulin secretory cell.So the cell of differentiation is the islets of langerhans precursor of hiding of the remnants in the pancreas conduit.A kind of embodiment comprises to described individuality to be used, and optimum decision system is used the gastrin/cck receptor part and the EGF receptors ligand of differentiation regeneration amount, and preferred EGF, as the EGF-51 that replaced use separately or combined administration.

Treatment of diabetes can also be by being transplanted to the islets of langerhans or the islets of langerhans precursor of purification need realize in patient's body of treatment.The cell that is used for transplanting normally obtains from donor pancreas, or from such as umbilical cord, the stem cell that obtains in the stem line of the cultivation of embryo or foundation.Described cell can for example, be expelled to pancreas, in kidney or the liver by transplanting such as the approach that is injected directly in the organ.In addition, described cell is used use by intravenous, for example, described cell is administered to portal vein or hepatic vein, for example, is expelled in the portal vein through liver by percutaneous.Can be by providing gastrin/cck receptor part and/or EGF receptors ligand that described cell is increased before transplanting or after transplanting to cell after transplanting or before transplanting and/or being divided into the functional islets cell.

If ex vivo differentiation islets of langerhans precursor, the pancreatic tissue that no matter is stem cell or outer planting can both partly or entirely be separated into isolated cells, be used for following differentiation step or amplification step, the human fetal pancreatic tissue transplantation that will stimulate like this is in the host mammal body then.

In the pancreatic tissue of outer planting with before the differentiation enhancing composition contact or the while, described cell mass, islets of langerhans precursor in the particularly described outer planting tissue can be under the situation that is with or without gastrin/cck receptor part, increase by the EGF receptors ligand that sufficient amount is provided, so that induce mitosis to take place.The optional at first pancreatic tissue of the described outer planting of enrichment in the islets of langerhans precursor, described cell is particularly expressed and islets of langerhans precursor or the relevant labelled protein of ductal epithelial cell, CK19 for example, nestin, CK7, CK8, CK18, carbonic anhydrase II, DU-PAN2, the cell of carbohydrate antigen 19-9 and mucin MUC1.The islets of langerhans precursor of immortalization can randomly prepare with the method that well known to a person skilled in the art, for example, and by transforming with hTERT.Described cell can be used the gastrin-receptor ligand stimulation then with the amplification of exsomatizing of EGF receptors ligand so that finish described atomization, before transplanting, in vivo or ex vivo differentiation become full ripe islet cells.

In another embodiment, gastrin/cck receptor ligand stimulation is to realize by chimeric insulin promoter-gastrin fusion gene construct that transgenic imports described precursor by expression.In another embodiment, the EGF receptors ligand stimulates by expressing through the intravital EGF receptors ligand of transgenic importing mammal gene to be realized.At USPN 5,434, provide the sequence of described EGF gene in 135.

In another embodiment, the stimulation of gastrin/cck receptor part and EGF receptors ligand realizes by coexpression (i) preprogastrin peptide precursor gene and the (ii) stable already intravital EGF receptors ligand of the described mammal gene that imports.

In another aspect of this invention, relate to the method that realizes mammiferous islets of langerhans precursor differentiation, comprise the described cell of combination of stimulation with gastrin/cck receptor part and EGF receptors ligand.In this preferred embodiment on the one hand, the gastrin stimulation is to realize by the stable expression that imports the intravital preprogastrin peptide precursor gene of described mammal.Described expression is subjected to the control of insulin promoter.The EGF receptors ligand, for example TGF-α stimulation imports the realization of the intravital EGF receptors ligand of described mammal expression of gene by the transgenic mode.In the further developing of said method, the stimulation of gastrin and TGF-α preferably is subjected to (i) preprogastrin peptide precursor gene and (ii) imports the influence of the co expression of the intravital EGF receptors ligand of described mammal.Can instruct the suitable promoter of described genetic transcription to comprise viral promotors and cell promoter.Viral promotors comprises immediately early stage cytomegalovirus (CMV) promoter (Boshart etc. (1985) Cell 41:521-530), SV40 promoter (Subramani etc. (1981) Mol.Cell.Biol.1:854-864) and from the major late promoter (Kaufman and Sharp (1982) Mol.Cell.Biol.2:1304-13199) of adenovirus 2.One of gastrin/cck receptor ligand gene and EGF receptors ligand gene or the two expression preferably are subjected to the control of insulin promoter.

Another aspect of the present invention is a nucleic acid construct.This construct comprises the nucleotide sequence and the insulin transcriptional regulatory sequences of coding preprogastrin peptide precursor, it be positioned at coding preprogastrin peptide precursor sequence 5 ' and effectively support transcribing of this sequence.Described insulin transcription regulating nucleotide sequence preferably includes at least one insulin promoter.In preferred embodiments, the nucleotide sequence of coding preprogastrin peptide precursor comprises the exon 2 that contains people's gastrin gene and 3 polynucleotide sequence, and randomly comprises introne 1 and 2.

Another embodiment of the present invention is an expression cassette, and it comprises (i) encoding mammalian EGF receptors ligand, for example, and the nucleotide sequence of TGF-α and its transcription regulating nucleotide sequence; (ii) the encode nucleotide sequence of preprogastrin peptide precursor and its transcription regulating nucleotide sequence.The preferably strong non-tissue-specific promoter of the described transcription regulating nucleotide sequence of EGF receptors ligand is as metallothionein promoter.The described transcription regulating nucleotide sequence of preprogastrin peptide precursor is an insulin promoter.The preferred form of this embodiment is such, and wherein, the nucleotide sequence of described coding preprogastrin peptide precursor comprises the introne 1 that contains people's gastrin gene and the polynucleotide sequence of 2 exon 2s and 3.

Another aspect of the present invention relates to the carrier that comprises following expression cassette, and this expression cassette comprises preprogastrin peptide precursor coded sequence.This carrier can be a plasmid, maybe can be phage as pGeml, and it has the transcription regulating nucleotide sequence that comprises insulin promoter.

Another aspect of the present invention relates to the compositions of carrier, and it comprises that (1) has encoding mammalian EGF receptors ligand, for example, the nucleotide sequence of TGF-α, this sequence is subjected to strong non-tissue-specific promoter, for example, metallothionein promoter; With the preprogastrin peptide precursor coded sequence that is subjected to insulin promoter.At this on the one hand, each carrier can be a plasmid, as plasmid pGeml or phage.In addition, described expression cassette or carrier can also insert the viral vector with suitable tissue parent preferendum.The example of viral vector comprises adenovirus, herpes simplex virus, adeno associated virus, retrovirus and slow virus etc.Referring to (1996) such as Blomer, Human MolecularGenetics 5Spec.No:1397-404; With (1998) Trends inBiotechnology 16:35-40 such as Robbins.Already adenovirus mediated gene therapy is successfully used to instantaneous correction and had suffered from the patient's of cystic fibrosis the chloride transhipment defective of nasal epithelium.Referring to (1993) Cell 75:207-216. such as Zabner

Another aspect of the present invention is inhuman mammal or mammalian tissues, comprises its cell, and this cell can be expressed the gene of the coding preprogastrin of stable integration.The another embodiment of this respect is inhuman mammal, and it can express (i) preprogastrin peptide precursor gene; And/or (ii) EGF receptors ligand, for example, TGF-α gene, this gene stably be incorporated into non-human mammal already, in mammalian tissues or the cell.Described mammalian tissues or cell can be that the people organizes or cell.

Treatment is used and compositions

Method of application includes, but are not limited to percutaneous, intramuscular, and intraperitoneal, intravenous, subcutaneous, intranasal, and oral route.Described chemical compound can be used by any conventional route, for example, by transfusion or the injection of medicine group, comprises by epidermis or mucocutaneous lining absorbing (for example, oral mucosa, rectum and intestinal mucosa etc.), and can use with the other biological activating agent.Use preferably systematic.

The present invention also provides Pharmaceutical composition.Described compositions comprises the therapeutic agent for the treatment of effective dose and carrier or excipient that can be medicinal.Described carrier includes, but are not limited to saline solution, buffered saline solution, glucose, water, glycerol, ethanol, and their combination.Described preparation should be fit to described method of application.Be used for carrier and preparation that can be medicinal of the present invention and be disclosed in following document: Remington ' s Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, PA, 17 ^ThEd. (1985), the document is done this paper reference by receipts.Relevant medicine send the brief overview of the method for passing, can be referring to following document: Langer (1990) Science 249:1527-1533, and the document is done this paper reference by receipts.

When preparation Pharmaceutical composition of the present invention, may need to change compositions of the present invention, so that change their pharmacokinetics and bio distribution.The general discussion of relevant pharmacokinetics, the same referring to Remingtons ' s Pharmaceutical Sciences, Chapters 37-39.Be used to change pharmacokinetics and chorologic several different methods and be those of ordinary skills' known (for example,, the same) referring to Langer.The example of described method is included in the vesicle the described reagent of protection, and described vesicle is by such as albumen, lipid (for example, liposome), and carbohydrate, or the material of synthetic polymer is formed.For example, described reagent of the present invention can be mixed in the liposome, so that strengthen their pharmacokinetics and bio distribution feature.There is several different methods to can be used for preparing liposome, for example, disclosed in the following document: Szoka etc. (1980) Ann.Rev.Biophys.Bioeng.9:467, U.S. Patent number 4,235,871,4,501,728 and 4,837,028, more than all documents all done this paper reference by receipts.It is known that various other send delivery system, and can be used for using therapeutic agent of the present invention, for example, and minitype particle and microencapsulation etc.

If desired, described compositions can also contain the wetting agent or the emulsifying agent of trace, or the pH buffer agent.Described compositions can be a liquid solution, suspension, emulsion, tablet, pill, capsule, slow releasing preparation, or powder.Described compositions suppository be can be mixed with, traditional binding agent and carrier be used, as triglyceride.Oral formulations can comprise standard vector, as the mannitol of pharmaceutical grades, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.

In preferred embodiments, described compositions is according to the conventional method preparation, as being fit to the Pharmaceutical composition of using in the human vein.Usually, being used for compositions that intravenous uses is solution with the solutions in sterile isotonic aqueous buffer preparation.If necessary, described compositions can also comprise stabilizing agent, and local anesthetic, so that alleviate any pain of injection site.Generally, described composition provides separately, or mixes with unit dosage forms and to provide, for example, as the powder of exsiccant frozen drying or do not have the aqueous concentrate form and be contained in the airtight container, in ampere bottle or sachette, the quality of lined out activity agent.If described compositions is to use by transfusion, it can distribute by sterile pharmaceutical grade water or brinish infusion bottle are housed.If described compositions is to use by injection, can provide the sterilized water that is used to inject or the saline that are loaded on the ampere bottle, so that can before using, mix described composition.

Therapeutic agent of the present invention can be with the form preparation of neutrality or salt.Can comprise the salt that forms by free amino for medicinal salt, as come from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, the salt of tartaric acid etc., and the salt that forms with free hydroxy-acid group, as come from sodium, potassium, ammonium, calcium and other bivalent cations, 2-aminopropane., triethylamine, the 2-ethyl amido alcohol, histidine, the salt of procaine etc.

The consumption that can effectively treat particular disorder or situation of therapeutic agent of the present invention depends on the character of described disease or situation, and can determine by standard clinical techniques.The definite dosage that uses in described preparation also depends on route of administration, and the seriousness of described disease or disease, and should be according to doctor's judgement and each patient's situation decision.But, the suitable dosage ranges that is used for intravenous administration for the EGF receptors ligand is generally the about 0.01-500 microgram of every kg body weight reactive compound, and for the gastrin-receptor part, every kg body weight is typically about 0.1-5000 microgram reactive compound.Effective dose can be calculated according to the dose-effect curve from external or animal model test system.Suppository generally includes the active component of 0.5 weight %-10 weight %; Oral formulations preferably includes the 10%-95% active component.

In gene therapy method of the present invention, transfection is by transcribing therapeutic or the acquisition of expression vector importing mammalian hosts in the body, described expression vector is as exposed DNA, compound with Lipid carriers, particularly compound with cation lipid carrier, or insertion viral vector, for example recombinant adenovirus.Described importing mammalian hosts can be any one in the some kinds of approach, comprises intravenous or peritoneal injection, in the trachea, and in the sheath, parenteral, intra-arterial, intranasal, intramuscular, the part, percutaneous is applied to any mucomembranous surface, cornea installation etc.Interested especially is that described therapeutic expression vector is imported in the hole or chamber of instrument for circulation of body fluid or importing health.Therefore, intravenous use with sheath in to use be valuable especially because described carrier can extensively distribute after according to above-mentioned route of administration, and aerosol-applied also can be used for importing body orifice or chamber.According to the position of route of administration and use, can fixed specific cell and the tissue of target.For example, lacking under the fixed situation of any specific target, can transfection along direction of flow of blood near the tissue of injection site.If the use Lipid carriers can be modified them, so that utilize the cell of oriented molecule with described complex guiding particular type.Therefore, can adopt the antibody or the part of special receptor or other cell surface proteins, target cell is combined with specific surface protein.

Any physiology can be gone up acceptable medium and be used to use described DNA, recombinant viral vector or Lipid carriers, according to route of administration, use as deionized water, saline, the saline solution of phosphoric acid buffer and be dissolved in glucose of 5% in the water etc., the carrier that is used for described Pharmaceutical composition as indicated above etc.In described preparation, can comprise other compositions, as cushion, stabilizing agent, Biocide etc.This constituents has a large amount of records in the literature, and there is no need specifically to disclose in this article.Any composition of accumulative diluent of described complex or diluent that may cause all should be avoided, and comprises high salt and chelating agen etc.

The amount of employed treatment carrier is the amount that is enough to be provided at the therapeutic expression in the destination organization.The therapeutic expression is to be enough to make blood sugar level to reduce expression to normal level.In addition, the dosage of employed nucleic acid carrier must be enough to produce in the body ideal transgene expression level in affected tissue.In described application media, can comprise other DNA sequence, as adenovirus VA gene, and with the common transfection of described interested gene.If desired, the existence of the gene of encoding adenovirus VA gene outcome can significantly strengthen from described expression cassette and transcribe and the translation of the mRNA that comes.

There is multiple factor can influence expression in the transfection tissue, and therefore can be used for changing expression, so that adapt to specific purpose.When the needs high level expression, all factors can be optimized, and when the more weak expression of needs, can change one or more parameters, so that reach ideal expression.For example, can surpass the treatment window, just can use to be lower than optimal conditions if highly express.

Can on mRNA level as indicated above, determine the level and the tissue of described recombinant gene expression, and/or on polypeptide or protein level, determine.Can carry out quantitatively gene outcome by measuring its biologic activity in tissue.For example, protein active can be measured by method of immunity mentioned above, measure by assay method biology such as blood glucose, or the gene outcome that is tested and appraised in the transfectional cell is measured, wherein use the immunostaining technology, survey as the reporter gene product in the expression cassette with the antibody of gene outcome as described in can specific recognition or as described in being present in.

Usually, described therapeutic expression cassette is not incorporated in patient's genome, and if necessary, described treatment can repeat at any time according to the result that will obtain.If repeat described treatment, can monitor the patient, so that guarantee described treatment there are not disadvantageous immunity or other reactions.

The present invention also provides the method that is used at amplification in vitro a group pancreatic cell.When from suitable donor, separating pancreas, isolated cell, and in growth in vitro.Employed cell is to obtain from the tissue sample from the mammal donor, and described donor comprises people's corpse, the source of the fetus of pig or other suitable pancreatic cell.If use the human cell, under possible condition, described cell and receiver are main histocompatibility couplings.The purification of described cell can be by finishing by gradient separations after isolating pancreas being carried out enzymatic (for example collagenase) digestion.Allow the cell of purification in containing the culture medium of enough nutrients, produce, so that make described cell survival, described culture medium also comprises the beta-cell proliferation of the sufficient amount that contains gastrin/cck receptor part and EGF receptors ligand and induces compositions, so that can form the insulin secretion pancreatic beta cell.According to the present invention, after stimulating, described insulin secretion pancreatic beta cell can directly increase in culture before in being transplanted to the patient's body that needs treatment, or can directly transplanting after inducing compositions-treated with beta-cell proliferation.

Implantation method comprises to be transplanted to the insulin secretion pancreas beta cell that is obtained in patient's body of needs treatment, and unites the immunosuppressant of use such as ciclosporin.Can also before transplanting, the insulin production cell be encapsulated in the semipermeable membrane.Described film allows insulin to secrete from the cell of sealing, and can prevent that again described cell is subjected to immune attack simultaneously.The quantity of the cell of transplanting is every kg of patient body weight 10,000-20,000 insulin production β cell according to estimates.In order to keep effective treatment quantity of insulin secretory cell, may need repeated embryo transfer.According to the present invention, can also offer the gastrin/cck receptor part and the EGF receptors ligand of described transplanting receiver q.s, so that induce the propagation of the plain secreted β of islet cells and islet cell.

The treatment of diabetes effect can be assessed by the following method.If possible, the biological efficacy and the clinical efficacy of described therapeutic scheme are assessed.For example, disease shows self by improving blood sugar content, therefore, for example, recovers normally can assess the biological efficacy of treatment by the blood glucose trend of observing assessment.Described clinical efficacy, i.e. whether the treatment of described potential effect is effective aspect the process that changes disease, may more be difficult to measure.Although the assessment of described biological efficacy is very helpful as the alternative terminal point of clinical efficacy, it is not deterministic.Therefore, after for example having carried out treatment time of 6 months, mensuration can provide the clinical endpoint of the regenerated index of beta cell, and the index of the clinical efficacy of described therapeutic scheme can be provided.

Compositions of the present invention can be used as test kit and provides, for use in one or more methods.The test kit that is used for genetic therapy generally includes the therapeutic DNA construct, and this construct can be the DNA that exposes, and is with or without mitochondrion targeting sequence peptide, or recombinant viral vector or compound with Lipid carriers.In addition, Lipid carriers can independently provide in the container, so that compound with the DNA that is provided.Described test kit comprises the compositions that contains potent agent, said composition can be concentrate (comprising cryodesiccated compositions), can further dilute before using, and said composition can also provide to use concentration, wherein, can comprise one or more dosage in the bottle.Usually, in described test kit, single dosage can provide by enough aseptic bottles, so that the doctor can directly use described bottle, wherein, described bottle has the reagent that needs quantity and concentration.When described bottle is equipped with the preparation that will directly use, do not need to be used for other reagent of described method usually.Relevant with described test kit can be by the management medicine or biology goods production, the description of the form of government organs' regulation of using or selling, this description has embodied and has obtained described production, use, or sales management mechanism is to being used for the human permission of using.

The following examples are with form of description, rather than finite form provides.

Embodiment

Method

The following embodiment that is provided is provided following method, except as otherwise noted.

Animal

Normal Wistar and Zucker rat can freely normally be taken food, and can freely contact the water source, and before each research of beginning, adapt to 1 time-of-week.After inducing diabetes 5-7 days, by intravenous injection, use freshly prepd streptozotocin with the dosage of 80mg/kg body weight, described rat is divided into group at random, so that carry out processing subsequently.In embodiment 1-4, in containing the physiological saline solution of 0.1%BSA reprovision TGF-α and rat stomach secretin.According to the predetermined processing scheme of difference research, every animal is accepted the independent peritoneal injection (4.0 μ g/kg body weight) of 1 TGF-α or gastrin every day or with 1: 1 (w/w) combination (8.0 total μ g/kg) or with the PBS 10 day time of injection.

Under specific pathogen-free domestic condition, raise female NOD mice, and intensive care, so that in untreated female NOD mice, obtain 98% onset diabetes rate.Monitor the diabetes progress by begin to detect glycosuria by FBG every morning in 10 ages in week.When glycosuria occurring, measure fasting blood glucose level (FBG), and continuous 2 days FBG＞6.6mmol/1 are defined as diabetes.Usually select diabetic mice to use for the NOD model (embodiment 7) of nearest outbreak in 14-18 week.For chronic NOD model, select diabetic mice to use in 25 weeks that (FBG content usually＞30mM) (embodiment 5 and 6) during ages usually.In embodiment 8 and 9, the treatment of female NOD-SCID (the serious immunodeficiency that merges of immunity competition) mice is carried out during age in week at mice 5-7.In embodiment 8-9, usually handle mice 6-8 time-of-week, after transplanting, will begin in a minute, comprise and use separately that dosage is the human mutant EGF with 51 amino acid residues of 30 μ g/kg, wherein being positioned at No. 51 locational residues is agedoite (being disclosed in the application number 10/000,840), people's gastrin analog h Gastrin1-17Leul5 with 30-1000 μ g/kg, by peritoneal injection (i.p.) injection, every day 2 times, be present in saline/phosphate buffer.

Blood glucose

The processing stage when finishing, with the animal overnight fasting, and carry out intravenous (i.v.) or the test of intraperitoneal (i.p.) glucose tolerance.Collection is from the blood sample of fasting object, and collected specimens on the different time points after injectable dextrose monohydrate.The blood sugar concentration of analytic sample, preparation is used for the peptide content by specificity radioimmunoassay method analyst para-insulin C then, if desired, described analysis with have negligible cross reactivity from the C peptide of mice.

Organize the insulin analysis

When each research finishes, put to death described animal, and take out pancreas and people's islet transplantation thing (if transplanting) and weigh.

Little biological living tissue is taken out at different representative position in pancreatic tissue, and horse back quick freezing in liquid nitrogen, so that carry out immunohistochemistry, albumen and insulin assay.The pancreas sample of quick freezing is thawed rapidly, in deionized water, carry out duplicate samples such as ultrasonic disruption and taking-up and carry out protein determination, and before carrying out insulin assay, homogenate is carried out acid/ethanol extraction, and calculate total islets of langerhans content by RIA.

Freezing and extract people's islet transplantation thing so that analyze insulin content by immune analysis method, or fixing in formalin, so that carry out histologic analysis.The people's islet transplantation thing that obtains for analysis extracts in acid/ethanol, so that analyze insulin content by immune analysis method.

Immunohistochemical analysis

Results people islet transplantation thing, and be separated into cell preparation, use respectively to insulin, glucagon, amylase and cytokeratin (CK) 7 and 19 special antibody carry out immunostaining to described cell preparation.Implement immunohistochemistry technology by the method that is disclosed in the following document: (Diabetes 49:1810-1818,2000) such as Suarez-Pinzon WL.The percentage ratio of each different cell type and be to determine by the microscope slide of adding up painted and numbering at tissue of transplanting and the insulin content in cell preparation, each microscope slide comprises at least 12 of each sample, 000 cell, and the counting of each microscope slide carries out at least three times to be repeated, and uses 100 times of immersion oil object lens at the bright field microscopically.At least 6,000 cell of every kind of preparation statistics, and with blind test numbering repetition 2 times.Before transplanting, the corresponding value in described counting and the transplanted cells preparation is compared.

People's islets of langerhans preparation and transplanting

Press hereinafter described method, from the pancreatic tissue of human donor, prepared people's islets of langerhans according to former disclosed mode.Islets of langerhans is isolating from the human pancreas of the organ donor of brain death, has obtained donor relatives' Informed Consent Form.The human Ethics Committee of hospital has ratified tissue and has obtained and testing program.In the donor body, take out pancreas and the method for separating islets of langerhans and be the Lakey JRT that carries out according to the method that is disclosed in the following document etc., (1988) Diabetes 37:413-420 such as (1999) Cell transplant 8:285-292 and RicordiC..

By under the scrotum, transplanting people's islet transplantation in non-diabetic NOD/ mice (being equivalent to 2000 islets of langerhans).Usually, people's donor pancreas is used to transplant about 12 mices of about 10-.

Embodiment 1

With the influence of handling in TGF-α and the gastrin body the intravital pancreas insulin content of normal rat

Become research to compare this experimental design with control animal (untreated), to using TGF-α, gastrin, or the influence of the intravital pancreas insulin content of non-diabetic animal crossed of the combined treatment of TGF-α and gastrin.The group (n=5) of normal Wistar rat is dispensed to one of following four processed group.

I group: TGF-α: reprovision recombined human TGF-α in containing the Sterile Saline of 0.1%BSA, and use 10 day time with 0.8 μ g/ days dosage i.p..

II group: gastrin: synthetic rat stomach secretin I is dissolved in very rare ammonium hydroxide, and with the Sterile Saline reprovision that contains 0.1%BSA.Dosage i.p. with 0.8 μ g/ days uses 10 day time.

III group: TGF-α+gastrin: the combination of above-mentioned preparation is used 10 day time with top given dosage level i.p..

The IV group: control animal is only accepted the i.p. 10 day time of injection of excipient.

When described search time finishes (10 days), put to death all animals, and take out the pancreas sample by the following method: take out 5 biological living tissue samples (1-2mg) of pancreatic tissue from the different representative position of every pancreas in rat, and horse back quick freezing in liquid nitrogen is so that analyze insulin content.In order to analyze the pancreas insulin content, the pancreas sample of described quick freezing is thawed rapidly, in distilled water, carry out ultrasonic disruption, and duplicate samples such as taking-up are carried out protein determination and acid/ethanol extraction, carry out insulin radioimmunoassay, RIA (Green etc., (1983) Diabetes 32:685-690) then.According to protein content pancreas insulin content value is proofreaied and correct, and finally be expressed as μ g insulin/mg pancreas albumen.The all values that calculates is a mean+/-SEM, and utilizes Si Shi 2-sample t-check assessment significance,statistical.

Table 1

Handle normal rat with TGF-α and gastrin

Handle the pancreas insulin content

(μ g insulin/mg albumen)

Contrast 20.6+/-6.0

TGF-α 30.4+/-7.4 ^＊

Gastrin 51.4+/-14.0 ^*

TGF-α+gastrin 60.6+/-8.7 ^{* *}

^*TGF-α and contrast, p=0.34;

^*Gastrin and contrast, p=0.11;

^{* *}The combination of TGF-α and gastrin, p=0.007.

Shown in top table 1, compare with control animal, the pancreas insulin content significantly improves (p=0.007) in the animal body that TGF-α+gastrin was handled; Compare with control animal, the pancreas insulin content has improved about 3 times.Above data have been supported following hypothesis: the combination results of TFG-α and gastrin the increase of functional islets beta cell volume, this increase has embodied the overall conditions of beta cell hypertrophy (quantity increase) rather than beta cell hypertrophy (volume of each beta cell increases).

Embodiment 2

With the influence of handling in the assembly of TGF-α and gastrin the intravital pancreas insulin content of diabetic animal

Whether the combination that this experimental design is become to measure TGF-α and gastrin can be brought up to the intravital pancreas insulin content of diabetic animal (streptozotocin (STZ) was handled) and normal (non-STZ handles) the suitable level of animal.

Normal Wistar rat is accepted 1 intravenous injection that dosage is the STZ of 80mg/Kg body weight.This STZ dosage estimates to guarantee that the animal of studying suffers from diabetes, and but, they maintain the beta cell amount that still reduces of function.Before using, STZ is dissolved in the ice-cold 10mM citrate buffer solution immediately.Monitor described animal every day; Show the diabetes that continue existence by glycosuria, and confirm by non-empty stomach blood sugar detection.Inducing diabetes after 1 week, by the following method rat is being divided at random two groups (n=6).

I group: TGF-α+gastrin: by treatment S TZ diabetes rat with the combination i.p. injection 1 time of recombined human TGF-α and synthetic rat stomach secretin 1; These two kinds of preparations are used 10 day time with 0.8 μ g/ days dosage.

II group: contrast: the STZ diabetes rat is only accepted the i.p. 10 day time of injection of excipient.

When described search time finishes, put to death all animals, and press embodiment 1 described method and gather pancreas sample and analysis; The result is shown in following table 2.

Table 2

With TGF-α and gastrin processing chain urea rhzomorph rat

Handle the pancreas insulin content

(μ g insulin/mg albumen)

Contrast (STZ is only arranged) 6.06 ± 2.1

STZ+TGF-α+gastrin 26.7 ± 8.9

It is successful inducing diabetes by STZ, and produced medium, but continue the hypertension of degree.Do not produce total insulin deficit disease, maintain function so that guarantee the animal of studying, but the beta cell amount that reduces.

Shown in top table 2, the pancreas insulin content of described contrast streptozotocin processing animal is lower than 1/3 (20.6 ± 6.0mg insulin/mg albumen is referring to top table 1) of normal rat, and it is the result who destroys beta cell by STZ.In the STZ animal with the combined treatment of TGF-α and gastrin, described pancreas insulin content is higher than four times of the animal content of only accepting STZ, and identical with the content of normal rat statistically (referring to, Shang Mian table 1 for example).

Embodiment 3

With the influence of handling in TGF-α and the gastrin body the intravital IPGTT of the inductive diabetic animal of STZ-

The combination or the PBS of i.p. injection TGF-α and gastrin handle the two groups of inductive diabetes Wistar rat of (average weight 103g) STZ (n=6/ group) 10 day times by every day.Measured the fasting glucose of all rats at the 0th, 6 and 10 day.In order to determine that this insulin is excretory and is that function is arranged, carried out the IPGTT test, at the 10th day, after one night of fasting, carried out the intraperitoneal glucose tolerance and tested (IPGTT).Before with the dosage i.p. injectable dextrose monohydrate of 2g/kg body weight and 30,60 and 120 minutes afterwards, obtain blood sample from the tail vein.Carry out blood sugar detection as stated above.In two seminar, be similarly, but compare that the rat that 30,60 and 120 minutes TFG α and gastrin were handled after the i.p. injectable dextrose monohydrate shows blood glucose value and reduced by 50% (Fig. 1) with control rats in the blood sugar level of 0 time.

Embodiment 4

TGF-α and gastrin commute are suffered from the weight increase of diabetic animal and the influence of insulin content

The Zucker rat that before the beginning obesity, approximately obtained 30 ages in days in 10-15 days.Outside the Zucker rat of easy trouble diabetes (genotype fa/fa, autosomal recessives fat and diabetes suddenly change), in this research, also comprise the brood son of thin type non-diabetic (genotype+/+), as mentioned below.Monitor the development of obesity and the diabetes of described rat every day by measuring body weight and blood glucose.Diabetes start from 45-50 days usually in the intravital outbreak of Zucker rat, and compare by the thin type contrast suitable with the age, confirm significantly improving of blood sugar level.

This research comprises 5 groups, and every group of 5 rats are as shown in table 3.1 group and 2 groups (thin type, non-diabetic) usefulness TGF-α and gastrin combination or PBS processing respectively in the from the 0th to 10 day.3,4 and 5 groups comprise fat, early diabetes Zucker rat, genotype fa/fa.Before the diabetes outbreak, accept the combination pretreatment of 15 days (from-15 days to the 0th day) for 3 groups, and last till diabetes outbreak 10 days again (from the 0th day to the 10th day) afterwards.The 4th group after diabetes outbreaks with 10 day time of combined treatment of TGF-α and gastrin, the 5th group with the identical time of PBS processing.When this research finishes, put to death described rat, and take out pancreas.Take out little biological living tissue from different typical positions, carry out albumen and insulin assay as stated above.

Only carrying out pretreatment, handling or, between these are organized, do not show significant difference with the weight increase of the obese diabetic Zucker rat of saline treatment (in the table 33,4 and 5 groups).What is interesting is and notice or even with TGF-α+gastrin long time treatment (25 days, 3 groups), to normal weight increase also not effect.In the limits of error, weight increase is identical in all groups.

To in fat Zucker rat, TGF-α+gastrin processing compare the influence of fasting glucose and PBS contrast accordingly.On an empty stomach blood sugar content significantly improved (4.0 ± 0.6 and 5.0 ± 0.2) first by the 15th day, and selected this time point as with TGF-α+gastrin or contrast the zero-time of the processing of carrying out 10 day time with PBS.Handle not significantly change fasting blood glucose level by TGF-α+gastrin processing or by PBS.No matter handle with somatomedin or with PBS, compare with obese animal, fasting blood sugar is all lower in lean type animal.Shown in the described result table 3 and Fig. 2 below.

Embodiment 5

With of the dose dependent influence of gastrin interior therapeutic to the intravital fasting glucose of NOD mice of suffering from chronic insulin-dependent diabetes

The purpose of this test is to determine whether gastrin self can prevent the generation and the death of serious hyperglycemia in suffering from the NOD mice of chronic insulin-dependent diabetes.The NOD mice that suffers from chronic insulin-dependent diabetes and keep insulinize is divided into different processed group, handles with following composition: (i) excipient (n=4); (ii) G 11 μ g/kg/ days, twice (n=4) of i.p. injection every day injected 28 day time, (iii) G 15 μ g/kg/ days, twice (n=4) of i.p. injection every day injected 28 day time, (iv) G 110 μ g/kg/ days, twice (n=4) of i.p. injection every day injected 28 day time.After beginning 14 days, stop insulinize with the G1 treatment.G1 is 17 amino acid whose gastrin analog, and this length is identical with the length of natural gastrin molecule, but contains No. 15 locational single amino acids changes that become leu from met.

The from the 0th to the 14th day, described animal was kept by insulinize, and the fasting glucose of all processed group (FBG) level keeps near the level at the 0th day record, and except the group of 10 μ g/kg/ days G1 processing, this group shows the reduction of FBG.After stopping insulinize the 28th, 14 day, all animals in described vehicle group all died from diabetic ketoacidosis (DKA), the injection of insulin because all mices all place one's entire reliance upon.But, all mices of handling 2 time-of-weeks of under the situation that does not have insulin to handle, having survived with G1.The fasting blood glucose level of the mice of handling with 1 μ g/kg/ days G1 keeps higher, but along with the dosage that strengthens G1, the corresponding reduction of fasting blood glucose level (being respectively 5 and 10 μ g/kg/ days).Referring to Fig. 4. above result shows, in chronic diabetes insulin-dependent NOD mice, under the situation that does not make insulinize, can significantly improve glucose control with the gastrin processing.

Embodiment 6

In suffering from the NOD mice of insulin-dependent diabetes, influence with the dose dependent of handling in the EGF body fasting glucose

The purpose of this experiment is to determine whether EGF can stop serious hypertension and dead generation and raising pancreas insulin content in suffering from the NOD mice body of chronic insulin-dependent diabetes.The NOD mice that will suffer from chronic insulin-dependent diabetes and keep insulinize is divided into different processed group, handles with following material: (i) excipient (n=4); (ii) El0.25 μ g/kg/ days, every day is by twice (n=4) of i.p. injection, inject 28 day time, (iii) El1 μ g/kg/ days, every day is by twice (n=4) of i.p. injection, inject 28 day time, (iv) El 3 μ g/kg/ days, every day was by twice (n=4) 28 day time of injection of i.p. injection.After beginning 14 days, stop insulinize with the E1 processing.E1 has 51 amino acid whose EGF analog.

The from the 0th to the 14th day, wherein, described animal was to keep insulinize, and for all groups of handling with E1, fasting serum glucose (FBG) water-glass reveals dose dependent to be reduced.After stopping insulinize the 28th, 14 day, all animals of vehicle group all died from diabetic ketoacidosis (DKA), the injection of insulin because all these mices all place one's entire reliance upon.On the contrary, do not having under the situation of insulin injection with all NOD mices that E1 handles 2 time-of-weeks of all having survived.In addition, the reduction of the FBG of E1 processed group keeps being stabilized on 2 all observed levels before, and except the group of 0.25 μ g/kg/ days E1 processing, FBG kept higher at the 28th day in this group.See Fig. 5.Above data show, in chronic diabetes insulin-dependent NOD mice, under the situation that does not make insulinize, with the control of EGF processing can significantly improvement glucose.

Embodiment 7

With gastrin or EGF the NOD mice of the diabetes of suffering from nearest outbreak is carried out handling in the body influence to fasting glucose and pancreas insulin content

The purpose of this experiment is to determine whether gastrin or EGF self can improve the diabetic conditions of the NOD mice of the diabetes with nearest outbreak.Monitor diabetes development (fasting glucose, the FBG＞6.6mmol/l) of non-obese diabetes (NOD) female mice.After the diabetes outbreak, with following mass treatment mice (i) excipient (n=4), (ii) E1 1 μ g/kg/ days, inject once (n=5) by i.p. every day, injects 14 day time, (iii) G1 1 μ/kg/ days, inject once (n=5) by i.p. every day, injects 14 day time.Mice is not accepted insulin-alternate process.Monitoring fasting blood glucose level and pancreas insulin level.E1 has 51 amino acid whose EGF analog, and G1 is the gastrin analog, and its length is identical with natural gastrin, but comprises the change of single amino acids on No. 15 positions.

Handle in the matched group at excipient, fasting glucose (FBG) level doubles after 35 days.The FBG level of the animal of handling with E1 or G1 keeps the value that writes down near when diabetes show effects (the 0th day), although there is the lasting destruction of islet cells in this animal model.By the islet cells new life that EGF or gastrin stimulate, compensated the destruction of described cell at least.Referring to Fig. 7. also measured the pancreas insulin level of all animals.At the 35th day, because the destruction of beta cell, the pancreas insulin level reduced for the contrast that excipient is handled, and showed significantly improving of pancreas insulin level and compare with described pretreatment values with the animal that E1 or G1 handle.Referring to Fig. 8.Originally studies confirm that in the NOD mice short-term process of handling with E1 or G1 (14 days) can improve the pancreas insulin content, and stops the progress of diabetic conditions after treatment stops at least in 3 time-of-weeks after diabetes outbreak recently.

Embodiment 8

It is intravital and with the sign of people's islet transplantation thing of handling in gastrin and the EGF body to be transplanted to mice

People's islets of langerhans (being about as much as 2000 islets of langerhans) is transplanted under the scrotum of mice, and is used the 1.5g/kg glucose by intravenous injection and stimulate as hyperglycemia.Gather blood sample, and analyze as stated above.Data show that (Fig. 8 A) blood sugar concentration time course is similar to the mice that EGF/ gastrin mice and excipient were handled.But, as the reaction that identical hyperglycemia is stimulated, the amount of the people C peptide that discharges in the blood plasma of the mice of EGF/ gastrin-handled is that more than 5 times of mice plasma content handled of excipient (are respectively 9.2 and 1.8nmoles/L/ minute, referring to Fig. 8 A and 8B), show that the insulin of described processing in stimulating people's islets of langerhans of transplanting is effective aspect synthetic.Above data also show, compare with the contrast that excipient was handled, and the amount of people's beta cell of functional engrafted is obviously more in the mice body that gastrin/EGF handled.

The insulin content of 8 all analyst's islet transplantation things after transplanting.Insulin content (each graft 1.34 ± 0.21 μ g, p＞0.02 with the islet transplantation thing of the mice of handling with excipient; Fig. 9) or with islet cells and islet (each graft is lower than 0.7 μ g) in advance compare, handle having significantly improved insulin content (each graft 2.42 ± 0.28 μ g) with gastrin/EGF.

The immunocytochemistry inspection of people's islet transplantation thing finds, the beta cell percentage ratio (19.6 ± 1.2% of the intravital islet transplantation thing of handling with excipient of mice; Figure 10) compare, gastrin/EGF handles the percentage ratio (29.7 ± 1.2%) that has improved observed beta cell in the islet transplantation thing.The sum (4.4 ± 0.2 * 10 of observed beta cell in the graft of handling mice from the EGF/ gastrin ⁶Beta cell) same apparently higher than observed quantity (2.6 ± 0.2 * 10 in the graft of the mice of handling from excipient ⁶Beta cell).Compare by graft with the mice of handling from excipient, analysis has found to react on the gastrin/cell percentage ratio of EGF processing and the increase (table 4) of quantity from the α cell of the expression glucagon in the graft of the mice of gastrin/EGF processing.In addition, in the graft that gastrin/EGF handled, the ratio of CK19 dyeing vessel cell has improved.This is important, because the CK19/20 vessel cell is believed to comprise precursor colony (Gmyr, V. etc., 2001, the Diabetes49:1671-80 that can produce islet cells during islet neogenesis; Gmyr, V etc., Cell Transplantation, 2001,10:109-121).Islets of langerhans comprises a group stem cell, and different estimations accounts for the 1/5-1/3 of the total cell quantity of islets of langerhans greatly.Table 4 also shows with gastrin/EGF compositions-treated the time, the percentage ratio of the cell of identifying brings up to about 84% from about 53% or 59%, this mainly is because the raising of β and α secretory cell percentage ratio has shown that gastrin/EGF processing has stimulated the differentiation of stem cells in the islets of langerhans to become insulin secretory cell.

Compare with islet cells and islet in advance, in 8 weeks after transplant, in the mice that gastrin/EGF and excipient were handled, all have the minimizing of acinous cell quantity.Generally speaking, with excipient handle (26%) and in advance transplanted tissue's (20%) compare, the cell in gastrin/EGF graft is formed mobile (62% islets of langerhans and the CK19 cell) that shows the differentiation of deflection islets of langerhans.

Handle the increase of having induced the positive beta cell of insulin in people's islets of langerhans that NOD-SCID mice body is implanted into gastrin/EGF.In Figure 11, recently the content in people's islet transplantation thing of the mice of using excipient is higher in people's islet transplantation thing of the mice of adopting gastrin/EGF treatment for the painted beta cell of top picture (data are from the cell of complete islet transplantation thing) expression insulin.Bottom picture (data are from isolating islet transplantation thing cell) shows, compare with the mice that excipient is handled, in the isolated cells preparation of the people's islet transplantation thing preparation that comes results in the mice body of handling with gastrin/EGF, obviously higher by the quantity of the detected insulin staining cell of immunoperoxidase.

Handle mice with gastrin/EGF, significantly improved the expression of the labelling of potential pancreatic, described labelling is the precursor transcription factor PDX1 (Figure 12 and 13) in the human pancreatic island cell.This albumen by pancreas and duodenum hox genes I (PDX-1) coding is being important aspect growth of adjusting pancreas and the islet cell function, and it can regulate and control the insulin gene expression.

Also observed the co of PDX1 and insulin expression, as shown in this two width of cloth figure.Above result shows that gastrin/EGF can induce PDX1 to express, and improves the intravital people's pancreatic of the NOD-SCID mice amount of implanting.

Embodiment 9

Gastrin/the EGF that uses low dosage can stimulate the growth of people's beta cell in the human tissue graft, and improves the insulin secretion reaction

Similar with the method among the top embodiment 8, with excipient or with gastrin/EGF (EGF, 30 μ g/kg/ days of low dosage, and gastrin, 30 μ g/kg/ days, handle 6 time-of-weeks, use 1 time by peritoneal injection every day) handle the NOD-SCID mice, and measure the insulin secretion reaction.

Be after the 1.5g/kg glucose stimulates as hyperglycemia, in the mice of handling, only to have observed the small improvement (Figure 13) of blood glucose toleration by the intravenous application dosage with gastrin/EGF.But, observed the remarkable improvement of insulin secretion reaction, compared, discharged in the mice plasma that gastrin/EGF handles that more people C-peptide embodied as blood plasma by the mice of handling with excipient.Therefore, even under the occasion than the gastrin of low dosage, gastrin/EGF handles the improvement of the insulin secretion reaction that has caused people's islet transplantation thing.

Embodiment 10

Transplant human stem cell and be divided into insulin secretory cell

The purpose of this experiment is to determine stem cell, for example, from the cell line of setting up, umbilical cord, or embryo's stem cell can be used for substituting the islet transplantation thing and is used to implant in the diabetics body, and by being divided into insulin secretory cell with gastrin/EGF processing.

From cell line, or be to obtain, and implant in each of a plurality of type i diabetes patients from closely-related newborn individual (child, cousinry, niece, or nephew) from the stem cell of umbilical cord.When repeating this embodiment first, stem cell is implanted under the scrotum in embodiment 1.The additive method of implanting in repetition subsequently comprises that intravenous uses, and for example, is administered to portal vein or hepatic vein.

Formed receiver's group, and the stem cell of using doses for the patient of each receiver's group, this dosage is equivalent to about 5 islets of langerhans (about 10 ⁷Cell), about 50 islets of langerhans (about 10 ⁸Cell), about 100 islets of langerhans (about 2 * 10 ⁸Cell), about 500 islets of langerhans (about 10 ⁹Cell), about 1000 islets of langerhans (about 2 * 10 ⁹Or about 2000 islets of langerhans (about 4 * 10 cell), ⁹Cell) quantity of stem cell in, the stem cell content of the islets of langerhans of use accounts for 25% of total cell quantity, or about 2 * 106 stem cell of each islets of langerhans (total roughly cell quantity of relevant each islets of langerhans is referring to table 4).

Each is transplanted receiver's group further be divided into matched group and the processed group of only using excipient (saline/phosphate buffer).The IRB examination in hospital committee clinical trial informed consent table of standard being provided for all patients, and agree may accept the part of the test of placebo as them.Processed group is accepted people's class methods of standard, is gastrin/EGF compositions of about 3 μ g/kgEGF 51N and about 100 μ g/kg h Gastrin 1-17Leu 15 by peritoneal injection dosage, every day 2 times, is present in the excipient.In all receiver's groups insulinize is continued about one-month period, then with less amount, for example approximately the common dose of 50%-about 80% provides, and carries out repeatedly monitoring every day and the record of blood insulin and glucose simultaneously.Use extra insulin for as required all patients,, and write down the haemoconcentration of all insulin doses and insulin and glucose so that keep normal blood glucose.

Endpoint determination shows, in gastrin/EGF processed group, compares with the quantity of the needed stem cell of matched group, and the more a spot of stem cell of initial application can provide enough insulins.

Embodiment 11

EGF (E1) and gastrin (G1) to isolating people's islets of langerhans of in culture, keeping/beta cell group's influence

The purpose of this experiment is to determine to handle whether can increase the beta cell of islets of langerhans group in external adding with EGF and gastrin, and by which type of mechanism increases.From people's donor pancreas (n=5), separate the islet cells preparation, and according to the licensed-in method preparation ((1988) Diabetes 37:413-420 such as Lakey JRT etc. (1999) Cell transplant 8:285-292 and Ricordi C.) that is used to prepare the human islets of langerhans of islet cell transplantation.With each culture dish 1 * 10 ⁶The concentration inoculation islet cells of cell, and in the MEM of serum-free culture medium self or replenished EGF (0.3 μ g/ml), gastrin (l.0 μ g/ml), or cultivate 4 time-of-weeks in the culture medium of EGF+ gastrin combination, and in culture, keep 4 time-of-weeks again.

Before handling, the groups of cells of the described islets of langerhans of measuring by the immunohistochemistry antibody staining becomes: 7 ± 2% glucagoies ⁺A cells, 23 ± 3% insulins ⁺Beta cell, 17 ± 2%CK7 ⁺Vessel cell, 6 ± 1%CK19 ⁺Vessel cell, 33 ± 2% amylase ⁺Acinous cell, and 11 ± 1% Vimentins ⁺Mesenchymal cell (meansigma methods ± SE, n=5 donor pancreas).After 4 weeks, by EGF+ gastrin (+128%, p＜0.001), and increased the beta cell amount by EGF (+77%, p＜0.01), and can or have EGF or the culture medium of gastrin (60%, p＜0.01) increase by gastrin (1%).

Under the situation of not adding EGF or gastrin, cultivate again after 4 weeks, in the islets of langerhans of EGF+ gastrin-processing, exist the continuation of beta cell amount to increase (+244%, p＜0.001) (Figure 14).The islets of langerhans preparation of EGF+ gastrin-handled also has CK19 ⁺The increase of vessel cell (+580%, p＜0.001) (Figure 15), at CK19 ⁺Exist the increase of islet transcription factor PDX-1 (before cultivating, not have PDX to express, and only after handling for 2 weeks, it is brought up to 82 ± 5%PDX-1 in the vessel cell simultaneously with the EGF+ gastrin ⁺) (Figure 16) the .EGF+ gastrin can also increase the percentage ratio of A cells in the islets of langerhans culture, and CK7 ⁺The percentage ratio of vessel cell and acinous cell descends.

Compare with the control cultures that excipient is handled, be total to stimulation process after 4 weeks with E1 and G1, the percentage ratio of beta cell significantly increases.At cancellation this two kinds of peptides after 4 weeks, the quantity of the positive beta cell of insulin further significantly increase (with handle the observed beta cell quantity of beginning compare be almost 4 times).Compare with the cell of only handling, in cell, observed and significantly improve with these two kinds of factors processing with E1 or G1.On the contrary, handle the beta cell group's of islets of langerhans minimizing observing excipient the 8th week.

This result of experiment shows, the combined therapy that carries out with EGF and gastrin is the external remarkable beta cell group who strengthens people's islets of langerhans.Even cancel described peptide after 4 weeks, the quantity of sophisticated beta cell also can increase gradually in the cell that stimulated altogether with EGF and gastrin already, shows that they have collaborative and long effect to the beta cell group.

In addition, find that EGF mainly can increase CK19 ⁺Vessel cell group (precursor), and CK19 in the gastrin chief leading cadre class islets of langerhans ⁺The PDX-1 of vessel cell expresses induces (Figure 16).Is being important by pancreas and duodenum hox genes 1 (PDX-1) encoded protein aspect growth of adjusting pancreas and the islet cell function.PDX-1 can regulate and control insulin gene and express, and specific expressed relevant with the islet cells of range gene.

More than find to coincide with our transgenic mice data, wherein, EGF receptors ligand self (TGF-α) can stimulate the ductule cell, and the existence of gastrin is that to finish the islet neogenesis process that starts by the EGF receptors ligand necessary.

Above strong hint as a result E1 and G1 can be used at the human islet cells preparation of amplification in vitro, so that further be transplanted in the diabetics body.

Diabetes are a kind of like this diseases, wherein, potential physiology's defective is the shortage of beta cell, and it is because the beta cell that causes of self-immunprocess destroys or owing to divide the result who causes that exhausts of potentiality from the beta cell that chronic stimulation caused of the high cyclical level of glucose.The latter has finally caused following situation, and at this moment, beta cell is regenerated and/or the impaired degree to the beta cell overall loss of renewal process, and has caused the reduction of pancreas insulin content simultaneously.Above result shows, the combination of TGF-α and gastrin can be used for the treatment of diabetes, comprises the generation that stimulates sophisticated beta cell, so that the insulin content of pancreas is returned to the non-diabetic level.

Top studies show that of reporting by using the gastrin/cck receptor part such as gastrin, and/or stimulates such as the EGF receptors ligand of TGF-α, activated islet cells new life completely again in mammalian body in the ductule epithelium that becomes the human pancreas.Reported the research of TGF-α and gastrin transgenic overexpression in the relevant pancreas, it has disclosed the pancreas gastrin in the developmental effect of islets of langerhans, and has shown that TGF-α and gastrin all work regulating the islets of langerhans development.Therefore, it is a kind of feasible method that is used for the treatment of diabetes that remaining multipotency pancreatic duct cell regeneration is divided into sophisticated insulin secretory cell, this is to realize by the combination of the multiple factor of therapeutic administration or compositions, and described combination or compositions can provide their expressed in situ in described pancreas.

The result who handles with TGF-α and gastrin in the Zucker of type 2 diabetes mellitus rat model shows that blood sugar level does not have marked difference between processed group and matched group, and this may embody the instantaneous hypoglycemic effect after long-time (18 hours) fasting.Immunohistochemistry discovered with control animal and compared, and the animal body of handling in TGF-α and gastrin contains the remarkable increase (Fig. 3) of individual cells kitchen range quantity of the cell of insulin.More than find to have confirmed the increase of single beta cell in the adult rat pancreas after handling with TGF-α and gastrin.What is interesting is that this single beta cell kitchen range is uncommon in adult (stimulation) pancreas in rat.More than find to have supported TGF-α and the therapeutical effect of gastrin in 1 type and type 2 diabetes mellitus, because treatment is newborn and duplicate at beta cell.

The present invention is not limited to the disclosed specific embodiments of this paper.The improvement that can draw by above explanation and accompanying drawing belongs to the scope of claims.

Quoted various documents in this article, the content of these documents is received with their integral form does this paper reference.

Claims (8)

1. stripped method that is used to obtain the compositions of functional ripe pancreas beta cell, described method comprises:

(a) provide one or more the precursor pancreas beta cell group who expresses among CK19, nestin, CK7, CK8, CK18, carbonic anhydrase II, DU-PAN2, carbohydrate antigen 19-9 and the mucin MUC1;

(b) with independent or handle precursor pancreas beta cell with the EGF receptors ligand of gastrin/cck receptor ligand combination, its consumption is enough to increase the number of precursor pancreas beta cell; With

(c) the precursor pancreas beta cell that increases with gastrin-receptor part and/or EGF receptors ligand treating number, its consumption is enough to realize that described precursor pancreas beta cell is divided into functional ripe pancreas beta cell, to obtain the compositions of functional ripe pancreas beta cell;

Wherein said method is end user's embryonic stem cell not.

2. the stripped method of claim 1, wherein said precursor pancreas beta cell or ripe pancreas beta cell partly or entirely are separated into and are suitable for being transplanted to the intravital isolated cells of host mammal.

3. the stripped method of claim 1, wherein said gastrin/cck receptor part is a gastrin.

4. the stripped method of claim 3, wherein said gastrin is people's gastrin 1-17/Leu15.

5. the stripped method of claim 3, wherein said gastrin is gastrin 34, gastrin 17 or gastrin 8.

6. the stripped method of claim 1 wherein in step (b) or (c), by expressing chimeric insulin promoter-gastrin fusion gene and/or EGF receptors ligand gene, imports precursor with gastrin/cck receptor part and/or EGF receptors ligand.

7. any one stripped method of claim 1-6, wherein said EGF receptors ligand is EGF1-53, EGF1-48, EGF1-49 or EGF1-52.

8. any one stripped method of claim 1-6, wherein said EGF receptors ligand is TGF-α 1-48, TGF-α 1-47, TGF-α 1-51, amphiregulin or poxvirus somatomedin.

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