2.3 Mechanisms of Pancreatic Stones Formation
Pancreatic stones are，in fact，intraductal calculi.According to the locations of stones，pancreatic stones mainly divided into main pancreatic duct and branch duct stones.Calculi in main pancreatic ducts is more common in the tropical chronic pancreatitis（TCP），while alcoholic chronic pancreatitis（ACP）is mostly characterized by the formation in pancreatic branch ducts of calculi（Pitchumoni 1984）.Pancreatic stone also includes the precalcified forms and the calcified forms.However，the mechanisms of different forms of pancreatic stone formation are almost the same，including protein precipitation and increased CaCO3 crystal growth in pancreatic juice.Nevertheless，it is still not clear that which protein is secreted abnormally causing protein precipitation.Current theories or hypothesis involved mainly were as follows.
2.3.1 Pancreatic Stone Protein
Human pancreatic stone protein（PSP）is one of the regeneration（reg）gene proteins，which is located on the short arm of chromosome 2 in 2p12 spans.There are five immunoreactive forms PSP S1-5 detected in pancreatic juice.PSP S2-5 corresponds to the four isoforms distinguishable by SDS-PAGE（Stewart 1989）.It was believed to inhibit spontaneous calcium carbonate precipitation from highly supersaturated solutions and was called“lithostathine”by some.PSP was detectable in almost all pancreatic stones，ranging widely from only a trace amount to 1.21%as a percentage of the stone weight.A wide range of percentages（ranging from 0.01 to 41.9%）of pancreatic stone protein in the total protein suggests that the mechanisms and protein components involved in the stone formation are multifactorial（Jin et al.2002）.Current hypothesis about PSP included stabilizing pancreatic juice hypothesis，calcium binding hypothesis and adsorption hypothesis.
Stabilizing Pancreatic Juice Hypothesis
Sarles and Bernard（1991）suggested that PSP would inhibit calcite crystal nucleation and growth in the pancreatic juice to prevent pancreatic stone formation.Bimmler et al.（1997）produced rat PSP in a baculovirus expression system and confirmed its calcite crystal inhibitor activity.They also found several organic and inorganic components of pancreatic juice（trypsinogen，phosphate）which had inhibitory activity on calcium carbonate crystal formation.Addadi and Weiner（1985）also reported PSP had an unspecific inhibition of calcite crystal growth.However，De Reggi et al.（1998）proposed that the inhibitory property was dut to a high concentration of Tris buffer.Therefore，PSP might affect stone formation，but the debate on lithostathine function remains.
Calcium Binding Hypothesis
Lohse and Kraemer（1984）found PSP had four equivalent and independent calcium binding sites by using radioactive 45Ca in equilibrium dialysis experiments.They thought that calcium binding can modify the physico-chemical characteristics of PSP，result in the formation of protein plugs，which could illustrate the presence of the protein in pancreatic stones.However，Multigner et al.（1986）observed the absence of calcium in the proteic core of some stones.Therefore，calcium binding sites on PSP may explain its inhibition of calcite nucleation，precipitation and crystal growth.
Adsorption Hypothesis
Gross and Caro（Gross et al.1985；De Caro et al.1979）found that PSP had a greater affinity for the crystal.Sarles and Bernard（1991）thought that PSP can adsorb on crystal surface.Geider et al.（1996）proposed that the adsorption of PSP to the crystal resulted in the modification of the crystal shape.De Reggi et al.（1998）observed that the quantities of adsorbed PSP and albumin per unit of surface were in the same range and the adsorption of PSP on calcite was not much higher than for an amorphous phase（glass）.Therefore，the adsorption of PSP on calcite does not interact specifically.
2.3.2 Lactoferrin(https://www.daowen.com)
Lactoferrin（LF）is a globular glycoprotein 80 kDa，which is widely represented in tears，saliva，nasal secretions and other secretory fluids.Which is also present in PMN.And some acinar cells also secret it.It also has many immunoregulatory effects including antimicrobial activity（bacteriocide，fungicide）and innate defense.
The concentration of Lactoferrin tends to increase in CP.Hayakawa et al.（1983）found the concentration of LF was much higher the protein plugs by analyzing the pancreatic stones obtained from the 13 patients with chronic calcified pancreatitis（Nagai and Ohtsubo 1984）.This suggested that lactoferrin may play an important role in early stage of protein plug formation in the pancreatic duct.
2.3.3 Trypsinogen
Trypsinogen is found in pancreatic juice，which is theprecursor form or zymogen of trypsin.Once it is activated by enteropeptidase，the trypsin can activate more trypsinogen into trypsin.Allan and White（1974）found intraductal pancreatic zymogens were activated in some patients with CP.Hayakawa et al.（1994）observed the immunoreactivity of human cationic trypsin in protein extracts from pancreatic stones in CP patients ranged from 0 to 42.3 ng/µg protein.They also observed that more densely immunoreactivity was presented in the center of the stones than in the concentric laminar layer of the periphery by using an immunogold technic SEM.Tympner（1981）found trypsin activity increased in pure pancreatic juice aspirated from CP patients.Renner et al.（1980）also found trypsinogen increased highly in in the pure pancreatic secretions in ACP patients.Therefore，we can concluded that hyperconcentration of pancreatic zymogens and proteins and activation of the trypsinogen were associated with pancreatic stone formation in early stage.
2.3.4 Osteopontin
Osteopontin is a 44-kDa glycosylated phosphoprotein，which is one of a group of noncollagenous bone matrix components.Recently，it has been reported that there is a relation between osteopontin expression and several diseases associated with calcification，such as atherosclerosis，breast cancer，meningioma and urinary stone formation.
Nakamura et al.（2002）found that osteopontin mRNA was detected in CP but not in normal pancreas specimens.In situ RT-PCR，they revealed that osteopontin was expressed in acinar or ductal cells in all 11 CCP patients，whereas in 5 of 9 CP cases without pancreatic stones osteopontin was not expressed in acinar or ductal cells.Therefore，acinar cells and ductal cells may secrete osteopontin，which can bind to protein plague and may play a crucial role in in pancreatic stone formation.
2.3.5 GP-2
GP-2（glycoprotein-2）is the most abundant protein of the zymogen granule membrane of the exocrine pancreas，which is linked to the membrane through a glycophophatidyl inositol（GPI）bond（Colomer et al.1994）.GP-2 was also shown to be homologous to Tamm-Horsfall protein，a GPI linked protein produced in kidney and excreted in urine，which is the major component of hyaline casts found in urine.
GP-2 is present in pancreatic juice and makes up 5-8%of unstimulated juice protein in the rat and pig.Stimulation with secretin has no effect on GP-2 output in pancreatic juice while stimulation with caerulein or carbachol increases secretion，but with a slower time course than the secretion of digestive enzymes.GP-2 is also present in ductal proteinaceous plugs found in chronic pancreatitis Freedman et al.（1994）examined the protein composition of intraductal plugs from patients with noncalcific chronic pancreatitis by SDS-PAGE and found GP-2 were both a reproducible constituent and enrichedwithin intraductal plugs.Therefore，GP2 may play a role in pancreatic plug formation，which is an important step in pancreatic stone formation.